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
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
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
- «
- ‹
- 1
- ›
- »