The heterologous expression and in vitro biochemical characterization of the Hsp70 escort protein 1 and mitochondrial Hsp70 partner proteins of the Trypanosoma brucei parasite and humans
- Authors: Mahlalela, Maduma Ernst
- Date: 2023-10-13
- Subjects: hsp-70 , Heat shock proteins , Molecular chaperones , African trypanosomiasis , Trypanosoma brucei , Kinetoplastida , Neglected tropical disease
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/431832 , vital:72807 , DOI 10.21504/10962/431832
- Description: The 70 kDa family of heat shock proteins (Hsp70) plays a central role in the maintenance of cellular proteostasis, with paralogues occurring in all the major compartments of the eukaryotic cell. Hsp70s act in conjunction with proteins known as co-chaperones, as part of the larger molecular chaperone network. In the mitochondrion, Hsp70 (mtHsp70) is responsible for the import of proteins synthesized in the cytosol, protein folding in the matrix and the maintenance of the iron-sulphur cluster. In human cells mtHsp70 (HSPA9) is also referred to as mortalin, as the knockdown of the protein leads to cell mortality. Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in animals. In the T. brucei parasite there are three identical mtHsp70 (TbmtHsp70) proteins that are produced, forming part of the Hsp70 machinery that is essential for parasite survival. In humans, the levels of HSPA9 are often elevated in non-communicable diseases such as cancer and neurodegeneration. Despite their vital cellular roles, mtHsp70s are characteristically prone to self-aggregation. The binding of the Hsp70 escort protein (Hep1) is required to prevent the aggregation of mtHsp70 proteins, enabling the proteins to function. In many non-communicable diseases, mtHsp70 and other molecular chaperones such as heat shock protein 90 (Hsp90) are being investigated as potential drug targets. Existing anti-trypanosomal drugs for treating sleeping sickness are toxic, having adverse side effects that are potentially lethal. Investigations into Hsp70s, and other molecular chaperones, form part of the research into the discovery of novel and efficacious therapeutics. This is the first study to characterise Hep1 and investigate its partnership with mtHsp70 in T. brucei. The overall aim of this study was to comparatively assess the T. brucei and human mtHsp70/Hep1 partnerships. The putative T. brucei Hep1 (TbHep1) orthologue was analysed in silico, and it was found to possess a zinc finger domain consisting of anti-parallel β-sheets that are characteristic of canonical Hep1 proteins, whilst the N-terminal domain was unstructured. Based on sequence analysis, the regions outside of the zinc finger domains lacked conservation. Despite the lack of sequence conservation, the N- and C-terminal regions of TbHep1 shared segments of similarity with Hep1 orthologues of other kinetoplastid and trypanosomal orthologues. The same held true for the N- and C-termini of human Hep1 (HsHep1) when compared to other Hep1 orthologues of mammalian origin. Biochemical analysis revealed TbmtHsp70 and HSPA9 to be prone to self-aggregation, which was reduced by co-expression with TbHep1 and HsHep1, respectively. Recently Hep1 proteins have been determined to be present in the cytosol. In this study, TbHep1 and HsHep1 also interacted with the cytosolic Hsp70s, HSPA1A and TbHsp70, by preventing their thermally induced aggregation and stimulating their ATPase activities. TbHep1 and HsHep1 also suppressed the thermally induced aggregation of the model substrates malate dehydrogenase and citrate synthase, independently of Hsp70. To date, only two Hep1 orthologues, HsHep1 and LbHep1, have been found to function in a similar manner to a J-protein co-chaperone by stimulating the ATPase activities of their partner mtHsp70 proteins. In this study, TbHep1 stimulated the ATPase activity of TbmtHsp70. HsHep1 also stimulated the ATPase activity of TbmtHsp70. However, the mechanism of action still needs to be determined. This study also explored the potential of the Hep1 orthologues to be functionally activated by oxidative stress, which is prevalent in mitochondria. The abilities of TbHep1 and HsHep1 to reduce the thermally induced aggregation of malate dehydrogenase were enhanced under oxidative conditions. Disrupting the function of Hep1 has been found to eventually lead to cell death, and given the critical role played by mtHsp70 in the cell, this partnership could be exploited as a potential drug target. In conclusion, this study demonstrated that TbHep1 and HsHep1 functionally interact with mtHsp70s, whilst also possessing independent chaperone activities that are also potentially influenced by the environmental redox state. , Thesis (PhD) -- Faculty of Science, Biotechnology Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Mahlalela, Maduma Ernst
- Date: 2023-10-13
- Subjects: hsp-70 , Heat shock proteins , Molecular chaperones , African trypanosomiasis , Trypanosoma brucei , Kinetoplastida , Neglected tropical disease
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/431832 , vital:72807 , DOI 10.21504/10962/431832
- Description: The 70 kDa family of heat shock proteins (Hsp70) plays a central role in the maintenance of cellular proteostasis, with paralogues occurring in all the major compartments of the eukaryotic cell. Hsp70s act in conjunction with proteins known as co-chaperones, as part of the larger molecular chaperone network. In the mitochondrion, Hsp70 (mtHsp70) is responsible for the import of proteins synthesized in the cytosol, protein folding in the matrix and the maintenance of the iron-sulphur cluster. In human cells mtHsp70 (HSPA9) is also referred to as mortalin, as the knockdown of the protein leads to cell mortality. Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in animals. In the T. brucei parasite there are three identical mtHsp70 (TbmtHsp70) proteins that are produced, forming part of the Hsp70 machinery that is essential for parasite survival. In humans, the levels of HSPA9 are often elevated in non-communicable diseases such as cancer and neurodegeneration. Despite their vital cellular roles, mtHsp70s are characteristically prone to self-aggregation. The binding of the Hsp70 escort protein (Hep1) is required to prevent the aggregation of mtHsp70 proteins, enabling the proteins to function. In many non-communicable diseases, mtHsp70 and other molecular chaperones such as heat shock protein 90 (Hsp90) are being investigated as potential drug targets. Existing anti-trypanosomal drugs for treating sleeping sickness are toxic, having adverse side effects that are potentially lethal. Investigations into Hsp70s, and other molecular chaperones, form part of the research into the discovery of novel and efficacious therapeutics. This is the first study to characterise Hep1 and investigate its partnership with mtHsp70 in T. brucei. The overall aim of this study was to comparatively assess the T. brucei and human mtHsp70/Hep1 partnerships. The putative T. brucei Hep1 (TbHep1) orthologue was analysed in silico, and it was found to possess a zinc finger domain consisting of anti-parallel β-sheets that are characteristic of canonical Hep1 proteins, whilst the N-terminal domain was unstructured. Based on sequence analysis, the regions outside of the zinc finger domains lacked conservation. Despite the lack of sequence conservation, the N- and C-terminal regions of TbHep1 shared segments of similarity with Hep1 orthologues of other kinetoplastid and trypanosomal orthologues. The same held true for the N- and C-termini of human Hep1 (HsHep1) when compared to other Hep1 orthologues of mammalian origin. Biochemical analysis revealed TbmtHsp70 and HSPA9 to be prone to self-aggregation, which was reduced by co-expression with TbHep1 and HsHep1, respectively. Recently Hep1 proteins have been determined to be present in the cytosol. In this study, TbHep1 and HsHep1 also interacted with the cytosolic Hsp70s, HSPA1A and TbHsp70, by preventing their thermally induced aggregation and stimulating their ATPase activities. TbHep1 and HsHep1 also suppressed the thermally induced aggregation of the model substrates malate dehydrogenase and citrate synthase, independently of Hsp70. To date, only two Hep1 orthologues, HsHep1 and LbHep1, have been found to function in a similar manner to a J-protein co-chaperone by stimulating the ATPase activities of their partner mtHsp70 proteins. In this study, TbHep1 stimulated the ATPase activity of TbmtHsp70. HsHep1 also stimulated the ATPase activity of TbmtHsp70. However, the mechanism of action still needs to be determined. This study also explored the potential of the Hep1 orthologues to be functionally activated by oxidative stress, which is prevalent in mitochondria. The abilities of TbHep1 and HsHep1 to reduce the thermally induced aggregation of malate dehydrogenase were enhanced under oxidative conditions. Disrupting the function of Hep1 has been found to eventually lead to cell death, and given the critical role played by mtHsp70 in the cell, this partnership could be exploited as a potential drug target. In conclusion, this study demonstrated that TbHep1 and HsHep1 functionally interact with mtHsp70s, whilst also possessing independent chaperone activities that are also potentially influenced by the environmental redox state. , Thesis (PhD) -- Faculty of Science, Biotechnology Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-10-13
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:
- Date Issued: 2023-03-31
- 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:
- Date Issued: 2023-03-31
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:
- Date Issued: 2023-03-29
- 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:
- Date Issued: 2023-03-29
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