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
- 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
Stress-inducible protein 1: a bioinformatic analysis of the human, mouse and yeast STI1 gene structure
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
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
In-silico analysis of Plasmodium falciparum Hop protein and its interactions with Hsp70 and Hsp90
- Authors: Clitheroe, Crystal-Leigh
- Date: 2013
- Subjects: Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3896 , http://hdl.handle.net/10962/d1003819 , Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Description: A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
- Full Text:
- Date Issued: 2013
- Authors: Clitheroe, Crystal-Leigh
- Date: 2013
- Subjects: Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3896 , http://hdl.handle.net/10962/d1003819 , Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Description: A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
- Full Text:
- Date Issued: 2013
The role of Hsp90 in the Wnt pathway of MCF7 breast cancer cells
- Authors: Cooper, Leanne Claire
- Date: 2011
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3985 , http://hdl.handle.net/10962/d1004044 , Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Description: Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.
- Full Text:
- Date Issued: 2011
- Authors: Cooper, Leanne Claire
- Date: 2011
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3985 , http://hdl.handle.net/10962/d1004044 , Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Description: Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.
- Full Text:
- Date Issued: 2011
Investigating assay formats for screening malaria Hsp90-Hop interaction inhibitors
- Authors: Derry, Leigh-Anne Tracy Kim
- Date: 2019
- Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63345 , vital:28395
- Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
- Full Text:
- Date Issued: 2019
- Authors: Derry, Leigh-Anne Tracy Kim
- Date: 2019
- Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63345 , vital:28395
- Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
- Full Text:
- Date Issued: 2019
Over-expression, purification and biochemical characterisation of trypanosomal heat shock protein
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
- Full Text:
- Date Issued: 2003
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
- Full Text:
- Date Issued: 2003
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
- 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
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.
- Full Text:
- Date Issued: 2011
- 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.
- Full Text:
- Date Issued: 2011
Characterisation of a plasmodium falciparum type II Hsp40 chaperone exported to the cytosol of infected erythrocytes
- Maphumulo, Philile Nompumelelo
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- Full Text:
- Date Issued: 2013
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- Full Text:
- Date Issued: 2013
Biochemical characterization of plasmodium falciparum heat shock protein 70
- Matambo, Tonderayi Sylvester
- Authors: Matambo, Tonderayi Sylvester
- Date: 2004
- Subjects: Plasmodium falciparum , Malaria -- Prevention , Protein folding , Proteins -- Purification , Heat shock proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4134 , http://hdl.handle.net/10962/d1015767
- Description: Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
- Full Text:
- Date Issued: 2004
- Authors: Matambo, Tonderayi Sylvester
- Date: 2004
- Subjects: Plasmodium falciparum , Malaria -- Prevention , Protein folding , Proteins -- Purification , Heat shock proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4134 , http://hdl.handle.net/10962/d1015767
- Description: Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
- Full Text:
- Date Issued: 2004
Characterisation of Human Hsj1a : an HSP40 molecular chaperone similar to Malarial Pfj4
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
Investigating the role of heat shock proteins (Hsps) 40, 70 and 90 in the life cycle of Theiler's murine encephalomyelitis virus (TMEV)
- Mutsvunguma, Lorraine Zvichapera
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
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
Targeting allosteric sites of Escherichia coli heat shock protein 70 for antibiotic development
- Authors: Okeke, Chiamaka Jessica
- Date: 2019
- Subjects: Heat shock proteins , Escherichia coli , Allosteric proteins , Antibiotics , Molecular chaperones , Ligands (Biochemistry) , Molecular dynamics , Principal components analysis , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/115998 , vital:34287
- Description: Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation.
- Full Text:
- Date Issued: 2019
- Authors: Okeke, Chiamaka Jessica
- Date: 2019
- Subjects: Heat shock proteins , Escherichia coli , Allosteric proteins , Antibiotics , Molecular chaperones , Ligands (Biochemistry) , Molecular dynamics , Principal components analysis , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/115998 , vital:34287
- Description: Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation.
- Full Text:
- Date Issued: 2019
In silico analysis of human Hsp90 for the identification of novel anti-cancer drug target sites and natural compound inhibitors
- Authors: Penkler, David Lawrence
- Date: 2015
- Subjects: Heat shock proteins , Cancer -- Treatment , Molecular chaperones , Homeostasis , Carcinogenesis , Chemotherapy , Ligand binding (Biochemistry) , Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4162 , http://hdl.handle.net/10962/d1018938
- Description: The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
- Full Text:
- Date Issued: 2015
- Authors: Penkler, David Lawrence
- Date: 2015
- Subjects: Heat shock proteins , Cancer -- Treatment , Molecular chaperones , Homeostasis , Carcinogenesis , Chemotherapy , Ligand binding (Biochemistry) , Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4162 , http://hdl.handle.net/10962/d1018938
- Description: The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
- Full Text:
- Date Issued: 2015
The potential roles of interactions between STAT3, Hsp90, and Hop in the maintenance of self-renewal in mouse embryonic stem cells
- Authors: Setati, Mokgadi Michael
- Date: 2008
- Subjects: Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3981 , http://hdl.handle.net/10962/d1004040 , Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Description: Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.
- Full Text:
- Date Issued: 2008
- Authors: Setati, Mokgadi Michael
- Date: 2008
- Subjects: Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3981 , http://hdl.handle.net/10962/d1004040 , Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Description: Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.
- Full Text:
- Date Issued: 2008
Development and optimisation of a novel Plasmodium falciparum Hsp90-Hop interaction assay
- Authors: Wambua, Lynn
- Date: 2018
- Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62626 , vital:28216
- Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
- Full Text:
- Date Issued: 2018
- Authors: Wambua, Lynn
- Date: 2018
- Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62626 , vital:28216
- Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
- Full Text:
- Date Issued: 2018
The role of Hsp90/Hsp70 organising protein (Hop) in the Proliferation, Survival and Migration of Breast Cancer Cells.
- Authors: Willmer, Tarryn
- Date: 2012
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4130 , http://hdl.handle.net/10962/d1015720
- Description: Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.
- Full Text:
- Date Issued: 2012
- Authors: Willmer, Tarryn
- Date: 2012
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4130 , http://hdl.handle.net/10962/d1015720
- Description: Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.
- Full Text:
- Date Issued: 2012
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