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
Isolation and characterization of genes encoding heat shock protein 70s (hsp 70s) from two species of the coelacanth, Latimeria chalumnae and Latimeria menadoensis
- Modisakeng, Keoagile William
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
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
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