The role of Stress Inducible Protein 1 (STI1) in the regulation of actin dynamics
- 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
- Full Text:
- Date Issued: 2015
- 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
- Full Text:
- Date Issued: 2015
Isolation, purification and partial characterisation of cancer procoagulant from placental amnion-chorion membranes and its role in angiogenesis inflammation and metastasis
- Authors: Krause, Jason
- Date: 2014
- Subjects: Coagulation , Amnion , Chorion , Metastasis , Inflammation , Neovascularization
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10350 , http://hdl.handle.net/10948/d1020897
- Description: Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.
- Full Text:
- Date Issued: 2014
- Authors: Krause, Jason
- Date: 2014
- Subjects: Coagulation , Amnion , Chorion , Metastasis , Inflammation , Neovascularization
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10350 , http://hdl.handle.net/10948/d1020897
- Description: Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.
- Full Text:
- Date Issued: 2014
The effects of extracellular and intracellular Hop on cell migration processes
- 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
- Full Text:
- Date Issued: 2014
- 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
- 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
- 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
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