Characterization of Trypanosoma brucei Sti1 and its interactions with Trypanosoma brucei Hsp83 and human Hsp90
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Trypanosoma brucei , Heat shock proteins , HSP90 , HSP83 , Molecular chaperones
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Trypanosoma brucei , Heat shock proteins , HSP90 , HSP83 , Molecular chaperones
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-03-31
The Role of HOP in Emerin-Mediated Nuclear Structure
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
Investigating the role of Hsp90 and LRP1 in FN matrix dynamics
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
Establishment of human OCT4 as a putative HSP90 client protein: a case for HSP90 chaperoning pluripotency
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
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
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
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