Analysis of the human HSP70-HSP90 organising protein (HOP) gene - characterisation of the promoter and identification of a novel isoform
- Authors: Mattison, Stacey
- Date: 2018
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62821 , vital:28296
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
Identification of SNPs within the CYP2A6 enzyme of TNBC cell lines and the resulting change in activity
- Authors: Dingle, Laura Margaret Kirkpatrick
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64349 , vital:28536
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2017
Regulation of cell biology by extracellular species of the Hsp90- Hsp70 organising protein (Hop)
- Authors: Höft, Maxine Allison
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59199 , vital:27465
- Description: Expected release date-April 2019
- Full Text:
- Date Issued: 2017
The relationship between OCT4 and an aggressive phenotype in triple negative breast cancer (TNBC)
- Authors: Jackson, Hayley Claire
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59209 , vital:27477
- Description: Expected release date-April 2019
- Full Text:
- Date Issued: 2017
Identification of potential novel roles for Hsp70/Hsp90 organising protein (Hop) using proteomic analysis in human cells
- Authors: Wingate, Ianthe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64758 , vital:28598
- Description: Expected release date-May 2018
- Full Text:
- Date Issued: 2016
Recombinant expression, purification and in vitro interaction analysis of HOP and RhoC
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
Biotechnology from bench to market: the design, scale-up and commercialisation strategy development of a disruptive bioprocess for potable ethanol production
- Authors: Dhanani, Karim Colin Hassan
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55863 , vital:26750
- Description: The capacity of research institutions to engage in technology transfer activities has important implications on both economic development and technological advancement. This thesis explores the developmental and commercialisation processes involved in the transfer of a potentially disruptive bioprocessing technology for beverage alcohol production. Ethanolic fermentation strategies are of interest due to their global economic importance and their potential to produce clean renewable fuels in the future. Currently used methods are both energetically wasteful and economically inefficient. To this end more effective bioprocessing methods and implementation strategies are required to enable commercially viable decentralised small-scale ethanol production. Perfusion reactors have a number of advantages over batch and other continuous fermentation strategies. This study aimed to develop and study the fermentative efficiency of a perfusion tower bioreactor system at the bench scale, and subsequently through a scale up process to a low level commercial capacity. An HPLC method was developed for the Simultaneous quantification of common fermentation analytes; this was used to determine bench scale fermentation efficacies over an operational period. At steady state the ethanol volumetric productivity of the bench scale bioreactor system was 3.40 g. L-1.h-1, the average yield of ethanol to consumed sugar was 0.467 g.g -1, with an average sugar conversion percentage of 96%. Results showed that the tower perfusion bioreactor was appropriate for high performance ethyl alcohol fermentations. This reactor design was then scaled up to pilot scale and then commercial scale ca pacity. Similar efficienCies were achieved with these larger systems. Based on the process performance data obtained, a commercialisation strategy was developed and market performance was projected. It was found that productivity rates per unit volume were favourable, and the bioreactor system was determined to be very cost effective for a decentralised ethanolic beverage manufacturing model.
- Full Text:
- Date Issued: 2015
Characterisation of the HSP70-HSP90 organising protein gene and its link to cancer
- Authors: Weeks, Stacey
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56006 , vital:26764
- Description: HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP.
- Full Text:
- Date Issued: 2015
Characterization of the co-chaperones of Hsp70 and Hsp90 in Trypanosoma brucei and their potential partnerships
- Authors: Mokoena, Fortunate
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54543 , vital:26583
- Description: African Trypanosomiasis, which is caused by Trypanosoma brucei, is one of the crippling agents of social and economic development in Africa. T. brucei cycles between the cold-blooded insect vector, the tsetse fly (Glossina spp), and warm-blooded mammalian hosts. T. brucei, T. cruzi and L. major are mammal infecting kinetoplastid parasites that are collectively referred to as TriTryps. These parasites experience extreme environments as they move between their warm-blooded mammalian hosts and cold-blooded insect vectors which trigger extensive morphological transformations during the life-cycle of the parasite. Molecular chaperones have been implicated in parasite differentiation. TriTryps display significant expansions and diversity in the gene complements encoding molecular chaperones, especially J-proteins. Generally, J-proteins function as co-chaperones of Hsp70s, forming part of vital protein homeostasis processes. Hsp70s show a high degree of conservation, while J-proteins appear to be an extreme case of taxonomic radiation. Although several studies have focused on the molecular and cell biology of Hsp70s in some kinetoplastid parasites, knowledge is still lacking pertaining to J-proteins and their partnerships with Hsp70s. This thesis focused on the classification of kinetoplastid Jproteins into the four types by examining the domain organizations using T. brucei as a guide. The potential partnership of J-proteins and Hsp70s were postulated based on predicted subcellular localization. Kinetoplastid parasites, particularly T. brucei, have evolved an expanded and specialized J-protein machinery, likely to be a consequence of an evolutionary fitness/trait to adapt to diverse environment present in hosts and vectors. These analyses will yield insight into the process of parasite differentiation as well as provide new leads for chemotherapeutic treatments. The presence of the STI1 mediated Hsp90 hetero-complex formation has not been confirmed in T. brucei. To this end, in silico and biochemical techniques were used to characterize the role of TbSTI1, as an adaptor protein of Hsp70 and Hsp90. Through domain architecture analysis, sequence alignments, phylogenetic analysis and three-dimensional structure prediction, TbSTI1 was demonstrated to be the most conserved TPR containing co-chaperone of Hsp70 and Hsp83 in T. brucei and also shown to be highly similar to its eukaryotic homologues. Recombinant TbSTI1 was overproduced and purified in E.coli cells and subsequently shown to associate with TcHsp70 in a concentration dependent manner and associate weakly with TbHsp70.4. TbSTI1 and TbHsp83 were also demonstrated to be expressed and upregulated upon exposure to heat shock at the bloodstream stage of parasite development. In conclusion, this study is the first to report the interaction of TbSTI1 with a chaperone. Interactions between TbSTI1 and Hsp70s were demonstrated and therefore, the formation of the hetero-complex is predicted based the similarity of TbSTI1 to other STI1 proteins.
- Full Text:
- Date Issued: 2015
Identification of novel SNPSTRs by 454 sequencing in Nguni and Sotho-Tswana populations
- Authors: Laurence, Jo-Anne Elizabeth
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55885 , vital:26752
- Description: DNA profiling is currently performed by analysis of the electropherogram that results following the amplification of a panel of Short Tandem Repeat (STR) loci. A need has arisen, however, for the development of a typing method that generates results which are compatible and comparable with existing databases, but that have a higher discrimination power by supplying sequence data as well as repeat-number data. Recent studies that explore these alternative typing methodologies have revealed the existence of a number of STR variants. There is, however, little information about the exact nature and prevalence of these sub-alleles. There have also been limited population studies of the genetic profiles of sub-Saharan African populations, despite the fact that evidence suggests that there is greater genetic structure and genetic diversity in these populations. In this study, a processing protocol for the generation of 454 sequencing-ready amplicons of vWA, D2S441, D3S1358, D13S317, D21S11 and D7S820 loci was developed. This protocol was applied to buccal swabs collected from 144 individuals of the Nguni and Sotho-Tswana population groups. A total of 145 485 reads were obtained from the sequencing of these amplicons, of which 97 400 and 48 085 reads were obtained for the Nguni and Sotho-Tswana populations respectively. The proportional representation for each locus ranged from 8-20%, and the allele calls and observed frequencies of these alleles suggested a high degree of relatedness between population groups. The sequencing results, furthermore, enabled the identification of a number of previously undescribed STR variants and SNPSTRs; with allele 13´ for D13S317 representing a SNP that may be predictive of Nguni-ancestry. The results also demonstrated the usefulness of next generation sequencing for increasing the number of discernible alleles for STR profiling.
- Full Text:
- Date Issued: 2015
The development of biological tools to aid in the genetic investigation of the black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros mitochondrial genomes
- Authors: Parsons, Michelle
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56059 , vital:26769
- Description: The black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros are found in South Africa. A decline in the populations of these species has resulted due to human activities such as habitat fragmentation and poaching. This has contributed to the loss of genetic diversity amongst the black and white rhinoceros. Conservation and anti-poaching efforts are needed to help maintain genetic diversity. These efforts could be improved through the development of non-invasive techniques to examine DNA from threatened animals. The aim of this research was to develop a molecular technique which would allow for the identification of the black and white rhinoceros and to develop a molecular technique which would allow for intraspecies genetic variation to be examined. DNA extractions were performed on matched faecal and tissue samples that were collected from two regions in South Africa. Polymerase chain reaction (PCR) primer sets were designed to investigate several regions of the rhinoceros mitochondrial genome. PCR optimisation was completed for the target regions. Sequencing was conducted on all final PCR products. The cytochrome c oxidase subunit 1 (COIi) gene allowed for the rhinoceros family to be identified. This region was digested with the HindIII restriction enzyme, which allowed for the specific identification of either the black or white rhinoceros. A subsequent region of the cytochrome c oxidase subunit 1 (COIii) as well as the D-loop, hypervariable regions (HV1 and HV2), cytochrome b (cytb) and 16s rRNA regions were investigated. These regions displayed potential for establishing geographic origin for black rhinoceros samples, whereas the D-loop and HV2 show potential for the white rhinoceros. The white rhinoceros displayed sequence variation in the HV2 and cytb region, while variation was observed in the COIi and HV1 for the black rhinoceros. All investigated target regions allowed for the rhinoceros family to be identified. The COI (COIi and COIii), HV2 and cytb regions allowed for the subspecies of rhinoceros to be identified, however the D-loop was not able to identify the white rhinoceros species. The 16s rRNA and HV1 regions allowed for the correct subspecies of rhinoceros to be identified, however as the primers were only compatible for the black rhinoceros therefore a subsequent investigation is required for the white rhinoceros. The establishment of this novel PCR based technique to identify white and black rhinoceros will allow for efficient species identification in wildlife forensic cases. A biological method was established to study intraspecies variation for the white and black rhinoceros; however the investigated target regions did not yield sufficient genetic variation. The core techniques developed in this study will be valuable for future studies that wish to investigate genetic variation in mammal species.
- Full Text:
- Date Issued: 2015
The effect of extracellular Hsp90β and TGF-β1 on colon cancer biology
- Authors: Perks, Tamarin
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55896 , vital:26753
- Description: The TGF-β signaling pathway is known to be one of the most commonly mutated pathways in human cancers, while Hsp90 is a bone fide drug target that is involved in regulating the conformation and activity of many oncoproteins. The role of intracellular Hsp90 in cancer has thus far been established and there is a growing link between extracellular Hsp90 and cancer metastasis, as well as the role of TGF-β in metastasis. This study aimed to analyse the interaction between Hsp90 (both intracellular and extracellular) and the TGF-β machinery in cancer cells, as well as to determine the effect of these proteins on cellular responses on the biology of cancer cells. This was achieved by studying the expression of Hsp90; TGF-βRII and TGF-β1 in cancer cell lines of various origins using flow cytometry, ELISA, and western blot analysis. The genetically paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and lymph node metastasis, respectively, were selected for further study due to differences in expression levels and activation of the TGF-β1 pathway. SW480 cells expressed double the level of TGF-βRII compared to SW620 cells, while SW620 expressed two times more extracellular TGF-β1 than SW480 cells. A direct interaction between TGF-β1 and Hsp90β was determined in vitro, and confirmed in vivo in SW620 cells. Growth, adhesion and migration were analysed in SW480 and SW620 cells. SW480 cells adhered significantly faster than SW620 cells, while SW620 cells had a greater rate of migration. Inhibiting the TGF-β pathway, specifically TGF-βRI, using SB 431542, as well as inhibiting Hsp90 with novobiocin, caused an increase in migration in SW480 cells. Only the addition of TGF-β1 in combination with Hsp90 as well as SB 431542 caused an increase in migration in SW620 cells. The canonical TGF-β1/TGF-βRI/TGF-βRII pathway may be constitutively active in SW620 cells and the inhibition of TGF-βRI may suggest an alternate pathway or receptor in both SW480 and SW620 cells.
- Full Text:
- Date Issued: 2015
Analysis of the interaction of Hsp90 with the extracellular matrix protein fibronectin (FN)
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014
Structural bioinformatics analysis of the Hsp40 and Hsp70 molecular chaperones from humans
- Authors: Adeyemi, Samson Adebowale
- Date: 2014
- Subjects: Structural bioinformatics , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Biomolecules
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4171 , http://hdl.handle.net/10962/d1020962
- Description: HSP70 is one of the most important families of molecular chaperone that regulate the folding and transport of client proteins in an ATP dependent manner. The ATPase activity of HSP70 is stimulated through an interaction with its family of HSP40 co-chaperones. There is evidence to suggest that specific partnerships occur between the different HSP40 and HSP70 isoforms. While some of the residues involved in the interaction are known, many of the residues governing the specificity of HSP40-HSP70 partnerships are not precisely defined. It is not currently possible to predict which HSP40 and HSP70 isoforms will interact. We attempted to use bioinformatics to identify residues involved in the specificity of the interaction between the J domain from HSP40 and the ATPase domain from the HSP70 isoforms from humans. A total of 49 HSP40 and 13 HSP70 sequences from humans were retrieved and used for subsequent analyses. The HSP40 J domains and HSP70 ATPase domains were extracted using python scripts and classified according to the subcellular localization of the proteins using localization prediction programs. Motif analysis was carried out using the full length HSP40 proteins and Multiple Sequence Alignment (MSA) was performed to identify conserved residues that may contribute to the J domain – ATPase domain interactions. Phylogenetic inference of the proteins was also performed in order to study their evolutionary relationship. Homology models of the J domains and ATPase domains were generated. The corresponding models were docked using HADDOCK server in order to analyze possible putative interactions between the partner proteins using the Protein Interactions Calculator (PIC). The level of residue conservation was found to be higher in Type I and II HSP40 than in Type III J proteins. While highly conserved residues on helixes II and III could play critical roles in J domain interactions with corresponding HSP70s, conserved residues on helixes I and IV seemed to be significant in keeping the J domain in its right orientation for functional interactions with HSP70s. Our results also showed that helixes II and III formed the interaction interface for binding to HSP70 ATPase domain as well as the linker residues. Finally, data based docking procedures, such as applied in this study, could be an effective method to investigate protein-protein interactions complex of biomolecules.
- Full Text:
- Date Issued: 2014
The characterization of DNAJC3: elucidating the function of the TPR domains
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/55874 , vital:26751
- Description: DNAJC3 is a novel member of the DNAJ family with two domains linked to co-chaperone functions, namely the tetratricopeptide repeat (TPR) and J domain. Out of the two domains, the TPR domains are the least characterized. Therefore, the aim of this study was to characterize and elucidate additional functions of DNAJC3 TPR domains through in silico, in vitro and ex vivo approaches. Through multiple sequence and structural alignment as well as electrostatic potential analysis, DNAJC3 TPR domain were found to be most similar to TPR-containing proteins with Hsp90 or Hsp70 independent functions. In vitro pull down assays illustrated that DNAJC3 TPR domains did not interact with either cytosolic Hsp90 and Hsp70 or Grp78 and Grp94 directly, however a potential indirect interaction with Grp94 and Hsp90 was observed in mammalian lysates, via pull down assays; suggesting the formation of a complex between the proteins mediated by a specific substrate. DNAJC3 TPR domains were found to bind indiscriminately to both native and heat denatured substrates in a dose dependent manner. DNAJC3 TPR domains bound to β-galactosidase with greater affinity than malate dehydrogenase (MDH), suggesting that DNAJC3 TPR domains might exhibit substrate specificity that has not been reported before. Preliminary ex vivo analysis of DNAJC3 in mammalian cells showed that induced stress conditions did not alter the cytosolic or endoplasmic reticulum (ER) localization, or levels of DNAJC3 protein, suggesting that the protein is not stress inducible. However, protein levels of DNAJC3 were dramatically reduced by Hsp90 inhibitor novobiocin at 500 μM. Transient knockdown DNAJC3 did not change the protein levels of either Grp78 or Grp94, but decreased the protein levels of Hsp70/Hsp90 organizing protein HOP. On the other hand, protein levels of DNAJC3 were increased in HOP depleted cells. In conclusion, this study was the first to experimentally demonstrate that DNAJC3 TPR domains do not interact directly with Hsp90, Hsp70, Grp78 or Grp94, and therefore DNAJC3 is unlikely to participate in traditional co-chaperone interactions with those proteins via its TPR domain. However, the J domain is known to interact with Grp78. The discovery that DNAJC3 TPR domains resemble that of TPR-containing proteins with functions independent of Hsp90 or Hsp70 suggests that DNAJC3 might link the Hsp70/Grp78 chaperone machinery to non co-chaperone related functions, which requires further analysis.
- Full Text:
- Date Issued: 2014
A role for heat shock protein 90 (Hsp90) in fibronectin matrix dynamics
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
- Full Text:
- Date Issued: 2013
The screening and characterisation of compounds for modulators of heat shock protein (Hsp90) in a breast cancer cell model
- Authors: Moyo, Buhle
- Date: 2013 , 2013-07-18
- Subjects: Heat shock proteins Breast -- Cancer Breast -- Cancer -- Chemotherapy Breast -- Cancer -- Treatment Cancer cells Naphthoquinone PQQ (Biochemistry)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4060 , http://hdl.handle.net/10962/d1004129
- Description: Breast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
Molecular chaperone expression and function in breast cancer and breast cancer stem cells
- Authors: Sterrenberg, Jason Neville
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells , Cancer cells
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4141 , http://hdl.handle.net/10962/d1016238
- Description: The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.
- Full Text:
- Date Issued: 2012
Expression of heat shock proteins on the plasma membrane of cancer cells : a potential multi-chaperone complex that mediates migration
- Authors: Kenyon, Amy
- Date: 2011 , 2011-03-29
- Subjects: Heat shock proteins , Protein folding , Molecular chaperones , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4122 , http://hdl.handle.net/10962/d1013362
- Description: Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.
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- Date Issued: 2011
Probing the biocompatibility of biomedical interfaces using the Quartz Crystal Microbalance with Dissipation
- Authors: Cromhout, Mary
- Date: 2011
- Subjects: Biomedical materials , Nanostructured materials , Biomedical engineering , Quartz crystal microbalances , Blood proteins , Nanoparticles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4102 , http://hdl.handle.net/10962/d1010660
- Description: The biomedical application of nanotechnology has come into the spotlight, with the promise of ‘personalised’ therapeutics that couple early diagnosis with targeted therapeutic activity. Due to the rapid growth of the biomedical applications of nanoparticles, along with the lack of understanding concerning their interactions with biomolecules, there is a pressing need for the development of standard methods directed at investigating the effect of introducing these unique particles into the human body. The central aim of this research is to establish a platform directed at assessing the biological fate of pioneering therapeutic particulate agents, such as metallophthalocyanines (MPcs) and multi-walled carbon nanotubes (FMWCNTs). In particular, we proposed, that Quartz Crystal Microbalance with Dissipation (QCM-D) technology may be employed to assess the composition of blood protein corona deposited on the therapeutic surface, and subsequently assess the biocompatibility of such particles. The proposed method of protein detection utilises the nanogram sensitivity of QCM-D technology to monitor highly specific antibody-antigen interactions. In particular those interactions which occur when probe antibodies are used to detect adsorbed blood proteins deposited on target particle-modified sensor surfaces. Protein detection analysis was directed toward identification of surface bound human serum albumin, complement factor C3c, and human plasma fibrinogen. Preliminary analysis of generic biomedical surfaces indicated human serum albumin demonstrates a higher binding affinity towards positively charged surfaces (i.e. cysteamine self-assembled monolayer), followed by hydrophobic surfaces. Detection of complement C3c, corresponded with literature, where lower levels were detected on negatively charged surfaces (i.e. mercapto undecanoic acid self-assembled monolayer), and higher levels of more hydrophobic surfaces (i.e. 11-amino undecane thiol self-assembled monolayer). Human plasma fibrinogen was observed to favour hydrophilic over hydrophobic self-assembled monolayer surfaces, which was in accordance with literature. Application of the proposed protein detection method for biocompatibility analysis of target therapeutic molecules, namely metallophthalocyanines and acid functionalised multi-walled carbon nanotubes, demonstrated a dependence on modified-surface film characteristics, such as surface charge and topography with regards to human serum albumin and human plasma fibrinogen analysis representing new insights into their potential biomolecular interactions The highest levels of detected human serum albumin and complement C3c were detected on the GePcSmix-modified surfaces. AlPcSmix-modified surfaces analysis suggested the highest levels of human plasma fibrinogen. Two methods of acid functionalisation were employed, using both nitric and sulphuric acid, and pure nitric acid. A general increase in detected human serum albumin, corresponding with an increase in functionalisation time, was observed. Complement C3c detection suggested an increase in deposited complement C3c, with increasing functionalisation time, when assessing nitric acid functionalised multi-walled carbon nanotubes, and a decrease, with increasing functionalisation time, when assessing nitric and sulphuric acid functionalised multi-walled carbon nanotubes. Analysis of human plasma fibrinogen was inconclusive, as were cytotoxicity experiments utilising MCF-7 cells in the presence of metallophthalocyanine complexes, raising simultaneously important considerations for their application and study. In the first such detailed examination of its kind it was concluded that the proposed method of protein detection, using QCM-D, allows for the rudimentary but rapid means of analysis of select protein corona deposited on particulate biomedical surfaces.
- Full Text:
- Date Issued: 2011