Investigating assay formats for screening malaria Hsp90-Hop interaction inhibitors
- Authors: Derry, Leigh-Anne Tracy Kim
- Date: 2019
- Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63345 , vital:28395
- Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
- Full Text:
- Authors: Derry, Leigh-Anne Tracy Kim
- Date: 2019
- Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63345 , vital:28395
- Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
- Full Text:
The development of high-throughput assays to screen for potential anticancer and antimalarial compounds that target ADP-ribosylation factor 6 and its signalling machineries
- Authors: Khan, Farrah Dilshaad
- Date: 2019
- Subjects: ADP-ribosylation , Proteins -- Metabolism , Nucleoproteins , Malaria -- Chemotherapy , Cancer -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92952 , vital:30810
- Description: ADP-ribosylation factors (Arfs) are small GTP-binding proteins that cycle between active GTP-bound forms and inactive GDP-bound forms. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ArfGEFs activate Arfs by mediating the exchange of GDP for GTP, while ArfGAPs terminate Arf function by stimulating the hydrolysis of the terminal phosphate group of GTP. Arf6 is a major regulator of endocytic trafficking and reorganization of the actin cytoskeleton in eukaryotic organisms. Owing to its participation in wide range of fundamentally distinct cellular processes, Arf6 may be a drug target for cancer and malaria amongst other diseases. As with cancer cells, rapid growth and viability of eukaryotic pathogens likely places a heavy burden on their endocytic pathways and a critical reliance on Arf6 activity. A putative malarial homolog of Arf6 (PfArf6) localises to numerous puncta along the periphery of the parasite in the mature trophozoite life stage of the parasite (T. Swart, MSc dissertation). Owing to highly inefficient parasite transfection procedures and a relative shortage of well described and validated parasite organelle markers, the possible functions of PfArf6 were explored using HeLa cells as a surrogate model for parasites by fluorescence microscopy of cells transfected with GFP-tagged PfArf6. Partial co-localisation was observed with the mammalian markers HsArf6 and LC3, which suggested possible roles in Arf6-dependent endocytosis and autophagy, respectively. While these possible roles are currently under investigation in parasites, an overall long-term goal which was initiated in this study was to determine whether PfArf6 is a valid drug target. To chemically validate PfArf6 as a drug target, a potent inhibitor needs to be identified. This requires the development of assays that may be employed for high-throughput screening of compound libraries. To support this goal, a novel plate-based assay was developed using human Arf6. The assay relies on the selective binding of an Arf effector protein domain (GGA3) fused to glutathione-S-transferase (GST), to His-tagged Arf6 immobilised on a nickel-coated plate. The assay format was developed and could robustly distinguish HsArf6-GDP (inactive) from HsArf6-GTP (active). Furthermore, it could be employed to detect the deactivation of Arf6 by ArfGAP1-stimualted GTP hydrolysis, but not Arf6 activation by ARNO-stimulated GDP/GTP exchange (ARNO is an ArfGEF). The ArfGAP1 deactivation assay was chemically validated using a known ArfGAP inhibitor, QS11. An improved assay was developed that employs JIP4 as an Arf6-specific binding partner instead of GGA3. In addition to superior performance, the alternative assay format could potentially be exploited for cancer drug discovery, since Arf6-JIP4 interaction has been implicated in cancer cell invasion and metastasis. Both assays may be employed to explore alternative ArfGEFs and ArfGAPs that act on Arf6 and contribute to the advancement of cancer. In parallel experiments, where development of PfArf6 assays was the focus, several issues arose. Firstly, we could not prepare GDP- and GTP-bound forms of PfArf6 since EDTA-mediated nucleotide exchange appeared to irreversibly destabilise the protein. However, PfArf6 activation (i.e. the preparation of PfArf6-GTP) was possible when mediated by ARNO and assessed by tryptophan fluorescence kinetic assays, suggesting that PfArf6 may be expressed in GDP-bound form in E. coli. As with human Arf6, ARNO-mediated GDP/GTP exchange on PfArf6 was not detectable in the immobilised PfArf6-GGA interaction GST assay format. However, a more sensitive assay was developed which relies on the use of nickel-horseradish peroxidase to detect the binding of His-tagged PfArf6 to JIP4-GST immobilised on glutathione plates and could detect ARNO-mediated PfArf6 activation. Since we could not prepare PfArf6-GTP (that did not rely on the presence of the ArfGEF, ARNO), malarial ArfGAP deactivation studies were conducted using PfArf1 instead of PfArf6 in the GGA-GST interaction assay. Both PfArfGAP1and PfArfGAP2 stimulated GTP hydrolysis by PfArf1, but only the former was inhibited by the standard human ArfGAP inhibitor, QS11. The development of these simple, cost-effective assays can be used in the high-throughput screening of novel anticancer and antimalarial compounds that target Arf signalling machineries. In theory, the assay could be extended as a tool to identify novel inhibitors of the multitude of Arfs, ArfGEFs and ArfGAPs originating from any organism and hence has broad clinical significance.
- Full Text:
- Authors: Khan, Farrah Dilshaad
- Date: 2019
- Subjects: ADP-ribosylation , Proteins -- Metabolism , Nucleoproteins , Malaria -- Chemotherapy , Cancer -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92952 , vital:30810
- Description: ADP-ribosylation factors (Arfs) are small GTP-binding proteins that cycle between active GTP-bound forms and inactive GDP-bound forms. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ArfGEFs activate Arfs by mediating the exchange of GDP for GTP, while ArfGAPs terminate Arf function by stimulating the hydrolysis of the terminal phosphate group of GTP. Arf6 is a major regulator of endocytic trafficking and reorganization of the actin cytoskeleton in eukaryotic organisms. Owing to its participation in wide range of fundamentally distinct cellular processes, Arf6 may be a drug target for cancer and malaria amongst other diseases. As with cancer cells, rapid growth and viability of eukaryotic pathogens likely places a heavy burden on their endocytic pathways and a critical reliance on Arf6 activity. A putative malarial homolog of Arf6 (PfArf6) localises to numerous puncta along the periphery of the parasite in the mature trophozoite life stage of the parasite (T. Swart, MSc dissertation). Owing to highly inefficient parasite transfection procedures and a relative shortage of well described and validated parasite organelle markers, the possible functions of PfArf6 were explored using HeLa cells as a surrogate model for parasites by fluorescence microscopy of cells transfected with GFP-tagged PfArf6. Partial co-localisation was observed with the mammalian markers HsArf6 and LC3, which suggested possible roles in Arf6-dependent endocytosis and autophagy, respectively. While these possible roles are currently under investigation in parasites, an overall long-term goal which was initiated in this study was to determine whether PfArf6 is a valid drug target. To chemically validate PfArf6 as a drug target, a potent inhibitor needs to be identified. This requires the development of assays that may be employed for high-throughput screening of compound libraries. To support this goal, a novel plate-based assay was developed using human Arf6. The assay relies on the selective binding of an Arf effector protein domain (GGA3) fused to glutathione-S-transferase (GST), to His-tagged Arf6 immobilised on a nickel-coated plate. The assay format was developed and could robustly distinguish HsArf6-GDP (inactive) from HsArf6-GTP (active). Furthermore, it could be employed to detect the deactivation of Arf6 by ArfGAP1-stimualted GTP hydrolysis, but not Arf6 activation by ARNO-stimulated GDP/GTP exchange (ARNO is an ArfGEF). The ArfGAP1 deactivation assay was chemically validated using a known ArfGAP inhibitor, QS11. An improved assay was developed that employs JIP4 as an Arf6-specific binding partner instead of GGA3. In addition to superior performance, the alternative assay format could potentially be exploited for cancer drug discovery, since Arf6-JIP4 interaction has been implicated in cancer cell invasion and metastasis. Both assays may be employed to explore alternative ArfGEFs and ArfGAPs that act on Arf6 and contribute to the advancement of cancer. In parallel experiments, where development of PfArf6 assays was the focus, several issues arose. Firstly, we could not prepare GDP- and GTP-bound forms of PfArf6 since EDTA-mediated nucleotide exchange appeared to irreversibly destabilise the protein. However, PfArf6 activation (i.e. the preparation of PfArf6-GTP) was possible when mediated by ARNO and assessed by tryptophan fluorescence kinetic assays, suggesting that PfArf6 may be expressed in GDP-bound form in E. coli. As with human Arf6, ARNO-mediated GDP/GTP exchange on PfArf6 was not detectable in the immobilised PfArf6-GGA interaction GST assay format. However, a more sensitive assay was developed which relies on the use of nickel-horseradish peroxidase to detect the binding of His-tagged PfArf6 to JIP4-GST immobilised on glutathione plates and could detect ARNO-mediated PfArf6 activation. Since we could not prepare PfArf6-GTP (that did not rely on the presence of the ArfGEF, ARNO), malarial ArfGAP deactivation studies were conducted using PfArf1 instead of PfArf6 in the GGA-GST interaction assay. Both PfArfGAP1and PfArfGAP2 stimulated GTP hydrolysis by PfArf1, but only the former was inhibited by the standard human ArfGAP inhibitor, QS11. The development of these simple, cost-effective assays can be used in the high-throughput screening of novel anticancer and antimalarial compounds that target Arf signalling machineries. In theory, the assay could be extended as a tool to identify novel inhibitors of the multitude of Arfs, ArfGEFs and ArfGAPs originating from any organism and hence has broad clinical significance.
- Full Text:
Development and optimisation of a novel Plasmodium falciparum Hsp90-Hop interaction assay
- Authors: Wambua, Lynn
- Date: 2018
- Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62626 , vital:28216
- Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
- Full Text:
- Authors: Wambua, Lynn
- Date: 2018
- Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62626 , vital:28216
- Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
- Full Text:
Comparative localization studies of P.falciparum ADP-ribosylation factor proteins in P.falciparum parasites and hela cells using GFP tagged constructs
- Authors: Swart, Tarryn
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3537 , vital:20519
- Description: Expected release date-December 2018
- Full Text:
- Authors: Swart, Tarryn
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3537 , vital:20519
- Description: Expected release date-December 2018
- Full Text:
Development of a high-throughput bioassay to determine the rate of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
An investigation of the role of mitochondrial STAT3 and modulation of Reactive Oxygen Species in adipocyte differentiation
- Authors: Kramer, Adam Hildyard
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54632 , vital:26595
- Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
- Full Text:
- Authors: Kramer, Adam Hildyard
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54632 , vital:26595
- Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
- Full Text:
The involvement of TRAP1 in the mitochondrial localization of STAT3 in mammalian cells
- Authors: Kadye, Rose
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55760 , vital:26731
- Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
- Full Text:
- Authors: Kadye, Rose
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55760 , vital:26731
- Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
- Full Text:
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