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:
- 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.
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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:
- 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.
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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:
- 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:
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