Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion
- Ziningwa, Tawanda, Achilonu, Ikechukwu, Hoppe, Heinrich C, Prinsloo, Earl, Dirr, Heinrich W, Shonhai, Addmore
- Authors: Ziningwa, Tawanda , Achilonu, Ikechukwu , Hoppe, Heinrich C , Prinsloo, Earl , Dirr, Heinrich W , Shonhai, Addmore
- Date: 2016
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431765 , vital:72802 , xlink:href="https://doi.org/10.1007/s12192-016-0678-4"
- Description: The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a Cterminal substrate binding domain (SBD). In the ADPbound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s.
- Full Text:
- Date Issued: 2016
- Authors: Ziningwa, Tawanda , Achilonu, Ikechukwu , Hoppe, Heinrich C , Prinsloo, Earl , Dirr, Heinrich W , Shonhai, Addmore
- Date: 2016
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431765 , vital:72802 , xlink:href="https://doi.org/10.1007/s12192-016-0678-4"
- Description: The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a Cterminal substrate binding domain (SBD). In the ADPbound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s.
- Full Text:
- Date Issued: 2016
Overexpression, Purification and Characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) Protein
- Zininga, Tawanda, Achilonu, Ikechukwu, Hoppe, Heinrich C, Prinsloo, Earl, Dirr, Heinrich W, Shonhai, Addmore
- Authors: Zininga, Tawanda , Achilonu, Ikechukwu , Hoppe, Heinrich C , Prinsloo, Earl , Dirr, Heinrich W , Shonhai, Addmore
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431719 , vital:72799 , xlink:href="https://doi.org/10.1371/journal.pone.0129445"
- Description: Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.
- Full Text:
- Date Issued: 2015
- Authors: Zininga, Tawanda , Achilonu, Ikechukwu , Hoppe, Heinrich C , Prinsloo, Earl , Dirr, Heinrich W , Shonhai, Addmore
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431719 , vital:72799 , xlink:href="https://doi.org/10.1371/journal.pone.0129445"
- Description: Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.
- Full Text:
- Date Issued: 2015
Plasmodium falciparum Hop (PfHop) interacts with the Hsp70 chaperone in a nucleotide-dependent fashion and exhibits ligand selectivity
- Zininga, Tawanda, Makumire, Stanley, Gitau, Grace W, Njunge, James M, Pooe, Ofentse J, Klimek, Hanna, Scheurr, Robina, Raifer, Hartmann, Prinsloo, Earl, Przyborski, Jude M, Hoppe, Heinrich C, Shonhai, Addmore
- Authors: Zininga, Tawanda , Makumire, Stanley , Gitau, Grace W , Njunge, James M , Pooe, Ofentse J , Klimek, Hanna , Scheurr, Robina , Raifer, Hartmann , Prinsloo, Earl , Przyborski, Jude M , Hoppe, Heinrich C , Shonhai, Addmore
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431752 , vital:72801 , xlink:href=" https://doi.org/10.1371/journal.pone.0135326"
- Description: Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses.
- Full Text:
- Date Issued: 2015
- Authors: Zininga, Tawanda , Makumire, Stanley , Gitau, Grace W , Njunge, James M , Pooe, Ofentse J , Klimek, Hanna , Scheurr, Robina , Raifer, Hartmann , Prinsloo, Earl , Przyborski, Jude M , Hoppe, Heinrich C , Shonhai, Addmore
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431752 , vital:72801 , xlink:href=" https://doi.org/10.1371/journal.pone.0135326"
- Description: Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses.
- Full Text:
- Date Issued: 2015
Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
- Full Text:
- Date Issued: 2007
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
- Full Text:
- Date Issued: 2007
The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum
- Shonhai, Addmore, Boshoff, Aileen, Blatch, Gregory L
- Authors: Shonhai, Addmore , Boshoff, Aileen , Blatch, Gregory L
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6486 , http://hdl.handle.net/10962/d1006269 , http://dx.doi.org/10.1110/ps.072918107
- Description: It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.
- Full Text:
- Date Issued: 2007
- Authors: Shonhai, Addmore , Boshoff, Aileen , Blatch, Gregory L
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6486 , http://hdl.handle.net/10962/d1006269 , http://dx.doi.org/10.1110/ps.072918107
- Description: It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.
- Full Text:
- Date Issued: 2007
Molecular chaperones in biology, medicine and protein biotechnology
- Boshoff, Aileen, Nicoll, William S, Hennessy, Fritha, Ludewig, M H, Daniel, Sheril, Modisakeng, Keoagile W, Shonhai, Addmore, McNamara, Caryn, Bradley, Graeme, Blatch, Gregory L
- Authors: Boshoff, Aileen , Nicoll, William S , Hennessy, Fritha , Ludewig, M H , Daniel, Sheril , Modisakeng, Keoagile W , Shonhai, Addmore , McNamara, Caryn , Bradley, Graeme , Blatch, Gregory L
- Date: 2004
- Language: English
- Type: Article
- Identifier: vital:6457 , http://hdl.handle.net/10962/d1004479
- Description: Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
- Full Text:
- Date Issued: 2004
- Authors: Boshoff, Aileen , Nicoll, William S , Hennessy, Fritha , Ludewig, M H , Daniel, Sheril , Modisakeng, Keoagile W , Shonhai, Addmore , McNamara, Caryn , Bradley, Graeme , Blatch, Gregory L
- Date: 2004
- Language: English
- Type: Article
- Identifier: vital:6457 , http://hdl.handle.net/10962/d1004479
- Description: Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
- Full Text:
- Date Issued: 2004
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