Characterization of Trypanosoma brucei Sti1 and its interactions with Trypanosoma brucei Hsp83 and human Hsp90
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Trypanosoma brucei , Heat shock proteins , HSP90 , HSP83 , Molecular chaperones
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Trypanosoma brucei , Heat shock proteins , HSP90 , HSP83 , Molecular chaperones
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
A Comparison of Mitochondrial Heat Shock Protein 70 and Hsp70 Escort Protein 1 Orthologues from Trypanosoma brucei and Homo sapiens
- Authors: Hand, Francis Bryan
- Date: 2023-03-29
- Subjects: Trypanosoma brucei , Heat shock proteins , Molecular chaperones , Transport protein , AlphaFold , Mitochondrial heat shock protein
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422281 , vital:71927
- Description: The causative agent of African trypanosomiasis, Trypanosoma brucei (T. brucei), has an expanded retinue of specialized heat shock proteins, which have been identified as crucial to the progression of the disease. These play a central role in disease progression and transmission through their involvement in cell-cycle pathways which bring about cell-cycle arrest and differentiation. Hsp70 proteins are essential for the maintenance of proteostasis in the cell. Mitochondrial Hsp70 (mtHsp70) is a highly conserved molecular chaperone required for both the translocation of nuclear encoded proteins across the two mitochondrial membranes and the subsequent folding of proteins in the matrix. The T. brucei genome encodes three copies of mtHsp70 which are 100% identical. MtHsp70 self-aggregates, a property unique to this isoform, and an Hsp70 escort protein (Hep1) is required to maintain the molecular chaperone in a soluble, functional state. This study aimed to compare the solubilizing interaction of Hep1 from T. brucei and Homo sapiens (H. sapien). The recently introduced Alphafold program was used to analyze the structures of mtHsp70 and Hep1 proteins and allowed observations of structures unavailable to other modelling techniques. The GVFEV motif found in the ATPase domain of mtHsp70s interacted with the linker region, resulting in aggregation, the Alphafold models produced indicated that the replacement of the lysine (K) residue within the KTFEV motif of DnaK (prokaryotic Hsp70) with Glycine (G), may abrogate bond formation between the motif and a region between lobe I and II of the ATPase domain. This may facilitate the aggregation reaction of mtHsp70 orthologues and provides a residue of interest for future studies. Both TbHep1 and HsHep1 reduced the thermal aggregation of TbmtHsp70 and mortalin (H. sapien mtHsp70) respectively, however, TbHep1 was ~ 15 % less effective than HsHep1 at higher concentrations (4 uM). TbHep1 itself appeared to be aggregation-prone when under conditions of thermal stress, Alphafold models suggest this may be due to an N-terminal α- helical structure not present in HsHep1. These results indicate that TbHep1 is functionally similar to HsHep1, however, the orthologue may operate in a unique manner which requires further investigation. , Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 2023
- Full Text:
- Authors: Hand, Francis Bryan
- Date: 2023-03-29
- Subjects: Trypanosoma brucei , Heat shock proteins , Molecular chaperones , Transport protein , AlphaFold , Mitochondrial heat shock protein
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422281 , vital:71927
- Description: The causative agent of African trypanosomiasis, Trypanosoma brucei (T. brucei), has an expanded retinue of specialized heat shock proteins, which have been identified as crucial to the progression of the disease. These play a central role in disease progression and transmission through their involvement in cell-cycle pathways which bring about cell-cycle arrest and differentiation. Hsp70 proteins are essential for the maintenance of proteostasis in the cell. Mitochondrial Hsp70 (mtHsp70) is a highly conserved molecular chaperone required for both the translocation of nuclear encoded proteins across the two mitochondrial membranes and the subsequent folding of proteins in the matrix. The T. brucei genome encodes three copies of mtHsp70 which are 100% identical. MtHsp70 self-aggregates, a property unique to this isoform, and an Hsp70 escort protein (Hep1) is required to maintain the molecular chaperone in a soluble, functional state. This study aimed to compare the solubilizing interaction of Hep1 from T. brucei and Homo sapiens (H. sapien). The recently introduced Alphafold program was used to analyze the structures of mtHsp70 and Hep1 proteins and allowed observations of structures unavailable to other modelling techniques. The GVFEV motif found in the ATPase domain of mtHsp70s interacted with the linker region, resulting in aggregation, the Alphafold models produced indicated that the replacement of the lysine (K) residue within the KTFEV motif of DnaK (prokaryotic Hsp70) with Glycine (G), may abrogate bond formation between the motif and a region between lobe I and II of the ATPase domain. This may facilitate the aggregation reaction of mtHsp70 orthologues and provides a residue of interest for future studies. Both TbHep1 and HsHep1 reduced the thermal aggregation of TbmtHsp70 and mortalin (H. sapien mtHsp70) respectively, however, TbHep1 was ~ 15 % less effective than HsHep1 at higher concentrations (4 uM). TbHep1 itself appeared to be aggregation-prone when under conditions of thermal stress, Alphafold models suggest this may be due to an N-terminal α- helical structure not present in HsHep1. These results indicate that TbHep1 is functionally similar to HsHep1, however, the orthologue may operate in a unique manner which requires further investigation. , Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 2023
- Full Text:
Biochemical characterisation and small molecule modulation of the interaction between two cytosolic Hsp70s from Trypanosoma brucei and potential co-chaperones
- Authors: Bentley, Stephen John
- Date: 2018
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/63402 , vital:28407
- Description: Expected release date-April 2019
- Full Text:
- Authors: Bentley, Stephen John
- Date: 2018
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/63402 , vital:28407
- Description: Expected release date-April 2019
- Full Text:
Characterization of the Mitochondrial Plasmodium falciparum heat shock protein 70
- Authors: Nyakundi, David Onchong’a
- Date: 2017
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/44449 , vital:25408 , https://doi.org/10.21504/10962/44448
- Description: Malaria remains a global health problem and accounts for many deaths and illnesses in subSaharan Africa. Plasmodium falciparum, the causative agent of the most fatal form of malaria, expresses a repertoire of heat shock proteins for cytoprotection, survival and pathogenesis. The parasite genome encodes six Hsp70 proteins found in various cell compartments. However, the putative parasite mitochondrial Hsp70 (PfHsp70-3) has not been investigated. The J-proteins, Pfj1 and PFF1415c, were proposed to function as co-chaperones of PfHsp70-3. The biochemical characterization of PfHsp70-3 was initially complicated by the fact that the protein was insoluble when expressed in E. coli cells. Various approaches to solubilize it resulted in inactive protein. A general characteristic of eukaryotic mitochondrial Hsp70s is their insolubility and their reliance on an Hsp70 escort protein (Hep) for solubility and ultimate functions. In this study, a putative Hep protein was identified in the genome of P. falciparum that is referred to as PfHep1. Coexpression of PfHep1 with PfHsp70-3 resulted in soluble and biochemically active PfHsp70-3. Size exclusion chromatography was employed to separate PfHsp70-3 from PfHep1 after coexpression. PfHep1 suppressed thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were also found to be essential for maintaining the functions of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Furthermore, PfHep1 did not stimulate the basal ATPase or increase refoldase activities of PfHsp70-3 hence displaying no co-chaperone roles. The full-length putative mitochondrial type I J protein, Pfj1, could not be produced in E.coli but a truncated protein containing the J-domain was produced which stimulated both the ATPase and refoldase activities of PfHsp70-3. Further, this study demonstrated that both PfHep1 and PfHsp70-3 localized to the mitochondrion in the erythrocytic stage of P. falciparum development thus confirming in silico predictions of their localization. Besides, PfHsp70-3 was expressed during all stages of the intraerythrocytic cycle of parasite development and was heat inducible. Generally, the data obtained in this study will enhance the existing knowledge on the biology of the parasite mitochondrial chaperone functions and open the possible avenue of drug targeting considering the specificity of PfHsp70- 3 and PfHep1 partnerships.
- Full Text:
- Authors: Nyakundi, David Onchong’a
- Date: 2017
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/44449 , vital:25408 , https://doi.org/10.21504/10962/44448
- Description: Malaria remains a global health problem and accounts for many deaths and illnesses in subSaharan Africa. Plasmodium falciparum, the causative agent of the most fatal form of malaria, expresses a repertoire of heat shock proteins for cytoprotection, survival and pathogenesis. The parasite genome encodes six Hsp70 proteins found in various cell compartments. However, the putative parasite mitochondrial Hsp70 (PfHsp70-3) has not been investigated. The J-proteins, Pfj1 and PFF1415c, were proposed to function as co-chaperones of PfHsp70-3. The biochemical characterization of PfHsp70-3 was initially complicated by the fact that the protein was insoluble when expressed in E. coli cells. Various approaches to solubilize it resulted in inactive protein. A general characteristic of eukaryotic mitochondrial Hsp70s is their insolubility and their reliance on an Hsp70 escort protein (Hep) for solubility and ultimate functions. In this study, a putative Hep protein was identified in the genome of P. falciparum that is referred to as PfHep1. Coexpression of PfHep1 with PfHsp70-3 resulted in soluble and biochemically active PfHsp70-3. Size exclusion chromatography was employed to separate PfHsp70-3 from PfHep1 after coexpression. PfHep1 suppressed thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were also found to be essential for maintaining the functions of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Furthermore, PfHep1 did not stimulate the basal ATPase or increase refoldase activities of PfHsp70-3 hence displaying no co-chaperone roles. The full-length putative mitochondrial type I J protein, Pfj1, could not be produced in E.coli but a truncated protein containing the J-domain was produced which stimulated both the ATPase and refoldase activities of PfHsp70-3. Further, this study demonstrated that both PfHep1 and PfHsp70-3 localized to the mitochondrion in the erythrocytic stage of P. falciparum development thus confirming in silico predictions of their localization. Besides, PfHsp70-3 was expressed during all stages of the intraerythrocytic cycle of parasite development and was heat inducible. Generally, the data obtained in this study will enhance the existing knowledge on the biology of the parasite mitochondrial chaperone functions and open the possible avenue of drug targeting considering the specificity of PfHsp70- 3 and PfHep1 partnerships.
- Full Text:
Characterization of the co-chaperones of Hsp70 and Hsp90 in Trypanosoma brucei and their potential partnerships
- Authors: Mokoena, Fortunate
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54543 , vital:26583
- Description: African Trypanosomiasis, which is caused by Trypanosoma brucei, is one of the crippling agents of social and economic development in Africa. T. brucei cycles between the cold-blooded insect vector, the tsetse fly (Glossina spp), and warm-blooded mammalian hosts. T. brucei, T. cruzi and L. major are mammal infecting kinetoplastid parasites that are collectively referred to as TriTryps. These parasites experience extreme environments as they move between their warm-blooded mammalian hosts and cold-blooded insect vectors which trigger extensive morphological transformations during the life-cycle of the parasite. Molecular chaperones have been implicated in parasite differentiation. TriTryps display significant expansions and diversity in the gene complements encoding molecular chaperones, especially J-proteins. Generally, J-proteins function as co-chaperones of Hsp70s, forming part of vital protein homeostasis processes. Hsp70s show a high degree of conservation, while J-proteins appear to be an extreme case of taxonomic radiation. Although several studies have focused on the molecular and cell biology of Hsp70s in some kinetoplastid parasites, knowledge is still lacking pertaining to J-proteins and their partnerships with Hsp70s. This thesis focused on the classification of kinetoplastid Jproteins into the four types by examining the domain organizations using T. brucei as a guide. The potential partnership of J-proteins and Hsp70s were postulated based on predicted subcellular localization. Kinetoplastid parasites, particularly T. brucei, have evolved an expanded and specialized J-protein machinery, likely to be a consequence of an evolutionary fitness/trait to adapt to diverse environment present in hosts and vectors. These analyses will yield insight into the process of parasite differentiation as well as provide new leads for chemotherapeutic treatments. The presence of the STI1 mediated Hsp90 hetero-complex formation has not been confirmed in T. brucei. To this end, in silico and biochemical techniques were used to characterize the role of TbSTI1, as an adaptor protein of Hsp70 and Hsp90. Through domain architecture analysis, sequence alignments, phylogenetic analysis and three-dimensional structure prediction, TbSTI1 was demonstrated to be the most conserved TPR containing co-chaperone of Hsp70 and Hsp83 in T. brucei and also shown to be highly similar to its eukaryotic homologues. Recombinant TbSTI1 was overproduced and purified in E.coli cells and subsequently shown to associate with TcHsp70 in a concentration dependent manner and associate weakly with TbHsp70.4. TbSTI1 and TbHsp83 were also demonstrated to be expressed and upregulated upon exposure to heat shock at the bloodstream stage of parasite development. In conclusion, this study is the first to report the interaction of TbSTI1 with a chaperone. Interactions between TbSTI1 and Hsp70s were demonstrated and therefore, the formation of the hetero-complex is predicted based the similarity of TbSTI1 to other STI1 proteins.
- Full Text:
- Authors: Mokoena, Fortunate
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54543 , vital:26583
- Description: African Trypanosomiasis, which is caused by Trypanosoma brucei, is one of the crippling agents of social and economic development in Africa. T. brucei cycles between the cold-blooded insect vector, the tsetse fly (Glossina spp), and warm-blooded mammalian hosts. T. brucei, T. cruzi and L. major are mammal infecting kinetoplastid parasites that are collectively referred to as TriTryps. These parasites experience extreme environments as they move between their warm-blooded mammalian hosts and cold-blooded insect vectors which trigger extensive morphological transformations during the life-cycle of the parasite. Molecular chaperones have been implicated in parasite differentiation. TriTryps display significant expansions and diversity in the gene complements encoding molecular chaperones, especially J-proteins. Generally, J-proteins function as co-chaperones of Hsp70s, forming part of vital protein homeostasis processes. Hsp70s show a high degree of conservation, while J-proteins appear to be an extreme case of taxonomic radiation. Although several studies have focused on the molecular and cell biology of Hsp70s in some kinetoplastid parasites, knowledge is still lacking pertaining to J-proteins and their partnerships with Hsp70s. This thesis focused on the classification of kinetoplastid Jproteins into the four types by examining the domain organizations using T. brucei as a guide. The potential partnership of J-proteins and Hsp70s were postulated based on predicted subcellular localization. Kinetoplastid parasites, particularly T. brucei, have evolved an expanded and specialized J-protein machinery, likely to be a consequence of an evolutionary fitness/trait to adapt to diverse environment present in hosts and vectors. These analyses will yield insight into the process of parasite differentiation as well as provide new leads for chemotherapeutic treatments. The presence of the STI1 mediated Hsp90 hetero-complex formation has not been confirmed in T. brucei. To this end, in silico and biochemical techniques were used to characterize the role of TbSTI1, as an adaptor protein of Hsp70 and Hsp90. Through domain architecture analysis, sequence alignments, phylogenetic analysis and three-dimensional structure prediction, TbSTI1 was demonstrated to be the most conserved TPR containing co-chaperone of Hsp70 and Hsp83 in T. brucei and also shown to be highly similar to its eukaryotic homologues. Recombinant TbSTI1 was overproduced and purified in E.coli cells and subsequently shown to associate with TcHsp70 in a concentration dependent manner and associate weakly with TbHsp70.4. TbSTI1 and TbHsp83 were also demonstrated to be expressed and upregulated upon exposure to heat shock at the bloodstream stage of parasite development. In conclusion, this study is the first to report the interaction of TbSTI1 with a chaperone. Interactions between TbSTI1 and Hsp70s were demonstrated and therefore, the formation of the hetero-complex is predicted based the similarity of TbSTI1 to other STI1 proteins.
- Full Text:
Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
The plasmodium falciparum exported Hsp40 co-chaperone, PFA0660w
- Authors: Daniyan, Michael Oluwatoyin
- Date: 2014
- Subjects: Molecular chaperones Heat shock proteins Proteins -- Analysis Proteins -- Structure Plasmodium Plasmodium falciparum Malaria -- Prevention -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4108 , http://hdl.handle.net/10962/d1011780
- Description: Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
- Full Text:
- Authors: Daniyan, Michael Oluwatoyin
- Date: 2014
- Subjects: Molecular chaperones Heat shock proteins Proteins -- Analysis Proteins -- Structure Plasmodium Plasmodium falciparum Malaria -- Prevention -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4108 , http://hdl.handle.net/10962/d1011780
- Description: Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
- Full Text:
Modulation of Plasmodium falciparum chaperones PfHsp70-1 and PfHsp70-x by small molecules
- Authors: Cockburn, Ingrid Louise
- Date: 2013
- Subjects: Plasmodium falciparum Heat shock proteins Molecular chaperones Homeostasis Protein folding Malaria Antimalarials Escherichia coli
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3887 , http://hdl.handle.net/10962/d1001747
- Description: The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.
- Full Text:
- Authors: Cockburn, Ingrid Louise
- Date: 2013
- Subjects: Plasmodium falciparum Heat shock proteins Molecular chaperones Homeostasis Protein folding Malaria Antimalarials Escherichia coli
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3887 , http://hdl.handle.net/10962/d1001747
- Description: The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.
- Full Text:
- «
- ‹
- 1
- ›
- »