Screening for small molecule modulators of Trypanosoma brucei Hsp70 chaperone activity based upon alcyonarian coral-derived natural products
- Andreassend, Sarah K, Bentley, Stephen, Blatch, Gregory L, Boshoff, Aileen, Keyzers, Robert A
- Authors: Andreassend, Sarah K , Bentley, Stephen , Blatch, Gregory L , Boshoff, Aileen , Keyzers, Robert A
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/426045 , vital:72309 , xlink:href="https://doi.org/10.3390/md18020081"
- Description: The Trypanosoma brucei Hsp70/J-protein machinery plays an essential role in survival, differentiation, and pathogenesis of the protozoan parasite, and is an emerging target against African Trypanosomiasis. This study evaluated a set of small molecules, inspired by the malonganenones and nuttingins, as modulators of the chaperone activity of the cytosolic heat inducible T. brucei Hsp70 and constitutive TbHsp70.4 proteins. The compounds were assessed for cytotoxicity on both the bloodstream form of T. b. brucei parasites and a mammalian cell line. The compounds were then investigated for their modulatory effect on the aggregation suppression and ATPase activities of the TbHsp70 proteins. A structure–activity relationship for the malonganenone-class of alkaloids is proposed based upon these results.
- Full Text:
- Date Issued: 2020
- Authors: Andreassend, Sarah K , Bentley, Stephen , Blatch, Gregory L , Boshoff, Aileen , Keyzers, Robert A
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/426045 , vital:72309 , xlink:href="https://doi.org/10.3390/md18020081"
- Description: The Trypanosoma brucei Hsp70/J-protein machinery plays an essential role in survival, differentiation, and pathogenesis of the protozoan parasite, and is an emerging target against African Trypanosomiasis. This study evaluated a set of small molecules, inspired by the malonganenones and nuttingins, as modulators of the chaperone activity of the cytosolic heat inducible T. brucei Hsp70 and constitutive TbHsp70.4 proteins. The compounds were assessed for cytotoxicity on both the bloodstream form of T. b. brucei parasites and a mammalian cell line. The compounds were then investigated for their modulatory effect on the aggregation suppression and ATPase activities of the TbHsp70 proteins. A structure–activity relationship for the malonganenone-class of alkaloids is proposed based upon these results.
- Full Text:
- Date Issued: 2020
Plasmodium falciparum Hep1 is required to prevent the self aggregation of PfHsp70-3
- Nyakundi, David O, Vuko, Loyiso A M, Bentley, Stephen J, Hoppe, Heinrich C, Blatch, Gregory L, Boshoff, Aileen
- Authors: Nyakundi, David O , Vuko, Loyiso A M , Bentley, Stephen J , Hoppe, Heinrich C , Blatch, Gregory L , Boshoff, Aileen
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66109 , vital:28903 , https://doi.org/10.1371/journal.pone.0156446
- Description: publisher version , The majority of mitochondrial proteins are encoded in the nucleus and need to be imported from the cytosol into the mitochondria, and molecular chaperones play a key role in the efficient translocation and proper folding of these proteins in the matrix. One such molecular chaperone is the eukaryotic mitochondrial heat shock protein 70 (Hsp70); however, it is prone to self-aggregation and requires the presence of an essential zinc-finger protein, Hsp70-escort protein 1 (Hep1), to maintain its structure and function. PfHsp70-3, the only Hsp70 predicted to localize in the mitochondria of P. falciparum, may also rely on a Hep1 orthologue to prevent self-aggregation. In this study, we identified a putative Hep1 orthologue in P. falciparum and co-expression of PfHsp70-3 and PfHep1 enhanced the solubility of PfHsp70-3. PfHep1 suppressed the 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 indeed essential for maintaining the function of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Soluble and functional PfHsp70-3, acquired by co-expression with PfHep-1, will facilitate the biochemical characterisation of this particular Hsp70 protein and its evaluation as a drug target for the treatment of malaria. , This work was funded by grants from the National Research Foundation (NRF); grant number 87663 and Deutsche Forschungsgemeinschaft (DFG); grant number LI 402/14-1. D.O.N. is the recipient of academic development and training funds from Mwenge Catholic University, Moshi, Tanzania. S.J.B. is the recipient of an NRF Doctoral Innovation Scholarship.
- Full Text:
- Date Issued: 2016
- Authors: Nyakundi, David O , Vuko, Loyiso A M , Bentley, Stephen J , Hoppe, Heinrich C , Blatch, Gregory L , Boshoff, Aileen
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66109 , vital:28903 , https://doi.org/10.1371/journal.pone.0156446
- Description: publisher version , The majority of mitochondrial proteins are encoded in the nucleus and need to be imported from the cytosol into the mitochondria, and molecular chaperones play a key role in the efficient translocation and proper folding of these proteins in the matrix. One such molecular chaperone is the eukaryotic mitochondrial heat shock protein 70 (Hsp70); however, it is prone to self-aggregation and requires the presence of an essential zinc-finger protein, Hsp70-escort protein 1 (Hep1), to maintain its structure and function. PfHsp70-3, the only Hsp70 predicted to localize in the mitochondria of P. falciparum, may also rely on a Hep1 orthologue to prevent self-aggregation. In this study, we identified a putative Hep1 orthologue in P. falciparum and co-expression of PfHsp70-3 and PfHep1 enhanced the solubility of PfHsp70-3. PfHep1 suppressed the 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 indeed essential for maintaining the function of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Soluble and functional PfHsp70-3, acquired by co-expression with PfHep-1, will facilitate the biochemical characterisation of this particular Hsp70 protein and its evaluation as a drug target for the treatment of malaria. , This work was funded by grants from the National Research Foundation (NRF); grant number 87663 and Deutsche Forschungsgemeinschaft (DFG); grant number LI 402/14-1. D.O.N. is the recipient of academic development and training funds from Mwenge Catholic University, Moshi, Tanzania. S.J.B. is the recipient of an NRF Doctoral Innovation Scholarship.
- Full Text:
- Date Issued: 2016
The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x
- Daniyan, Michael O, Boshoff, Aileen, Prinsloo, Earl, Pesce, Eva-Rachele, Blatch, Gregory L
- Authors: Daniyan, Michael O , Boshoff, Aileen , Prinsloo, Earl , Pesce, Eva-Rachele , Blatch, Gregory L
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66098 , vital:28901 , https://doi.org/10.1371/journal.pone.0148517
- Description: publisher version , Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria. , This work was supported by grants from the National Research Foundation (NRF) and Medical Research Council (MRC) of South Africa. The ProteOn XPR36 IAS was purchased from a National Nanotechnology Equipment Programme grant from the Department of Science and Technology and the NRF of South Africa. Michael O. Daniyan was a recipient of the Education Trust Fund (ETF) Academic Staff Training and Development (AST and D) scholarship of Obafemi Awolowo University, Ile-Ife, Nigeria and a Rhodes University Council research bursary
- Full Text:
- Date Issued: 2016
- Authors: Daniyan, Michael O , Boshoff, Aileen , Prinsloo, Earl , Pesce, Eva-Rachele , Blatch, Gregory L
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66098 , vital:28901 , https://doi.org/10.1371/journal.pone.0148517
- Description: publisher version , Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria. , This work was supported by grants from the National Research Foundation (NRF) and Medical Research Council (MRC) of South Africa. The ProteOn XPR36 IAS was purchased from a National Nanotechnology Equipment Programme grant from the Department of Science and Technology and the NRF of South Africa. Michael O. Daniyan was a recipient of the Education Trust Fund (ETF) Academic Staff Training and Development (AST and D) scholarship of Obafemi Awolowo University, Ile-Ife, Nigeria and a Rhodes University Council research bursary
- Full Text:
- Date Issued: 2016
PFB0595w is a Plasmodium falciparum J protein that co-localizes with PfHsp70-1 and can stimulate its in vitro ATP hydrolysis activity
- Njunge, James M, Mandal, Pradipta, Przyborski, Jude M, Boshoff, Aileen, Pesce, Eva-Rachele, Blatch, Gregory L
- Authors: Njunge, James M , Mandal, Pradipta , Przyborski, Jude M , Boshoff, Aileen , Pesce, Eva-Rachele , Blatch, Gregory L
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431739 , vital:72800 , xlink:href="https://doi.org/10.1016/j.biocel.2015.02.008"
- Description: Heat shock proteins, many of which function as molecular chaperones, play important roles in the lifecycle and pathogenesis of the malaria parasite, Plasmodium falciparum. The P. falciparum heat shock protein 70 (PfHsp70) family of chaperones is potentially regulated by a large complement of J proteins that localize to various intracellular compartments including the infected erythrocyte cytosol. While PfHsp70-1 has been shown to be an abundant cytosolic chaperone, its regulation by J proteins is poorly understood. In this study, we characterized the J protein PFB0595w, a homologue of the well-studied yeast cytosolic J protein, Sis1. PFB0595w, similarly to PfHsp70-1, was localized to the parasite cytosol and its expression was upregulated by heat shock. Additionally, recombinant PFB0595w was shown to be dimeric and to stimulate the in vitro ATPase activity of PfHsp70-1. Overall, the expression, localization and biochemical data for PFB0595w suggest that it may function as a cochaperone of PfHsp70-1, and advances current knowledge on the chaperone machinery of the parasite.
- Full Text:
- Date Issued: 2015
- Authors: Njunge, James M , Mandal, Pradipta , Przyborski, Jude M , Boshoff, Aileen , Pesce, Eva-Rachele , Blatch, Gregory L
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431739 , vital:72800 , xlink:href="https://doi.org/10.1016/j.biocel.2015.02.008"
- Description: Heat shock proteins, many of which function as molecular chaperones, play important roles in the lifecycle and pathogenesis of the malaria parasite, Plasmodium falciparum. The P. falciparum heat shock protein 70 (PfHsp70) family of chaperones is potentially regulated by a large complement of J proteins that localize to various intracellular compartments including the infected erythrocyte cytosol. While PfHsp70-1 has been shown to be an abundant cytosolic chaperone, its regulation by J proteins is poorly understood. In this study, we characterized the J protein PFB0595w, a homologue of the well-studied yeast cytosolic J protein, Sis1. PFB0595w, similarly to PfHsp70-1, was localized to the parasite cytosol and its expression was upregulated by heat shock. Additionally, recombinant PFB0595w was shown to be dimeric and to stimulate the in vitro ATPase activity of PfHsp70-1. Overall, the expression, localization and biochemical data for PFB0595w suggest that it may function as a cochaperone of PfHsp70-1, and advances current knowledge on the chaperone machinery of the parasite.
- Full Text:
- Date Issued: 2015
Trypanosoma brucei J protein 2 is a stress inducible and essential Hsp40
- Ludewig, Michael H, Boshoff, Aileen, Horn, David, Blatch, Gregory L
- Authors: Ludewig, Michael H , Boshoff, Aileen , Horn, David , Blatch, Gregory L
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431798 , vital:72804 , xlink:href="https://doi.org/10.1016/j.biocel.2014.12.016"
- Description: Hsp40 proteins (also known as DnaJ or J proteins) serve as co-chaperones for Hsp70, but also display evidence of independent chaperone function. Furthermore, certain Hsp40s have been shown to be stress-inducible and essential. Trypanosomatids display a remarkable diversification of Hsp40 proteins, with numerous distinct Hsp40-like proteins encoded in the Trypanosoma brucei genome. This study investigated the role of one of the six T. brucei Type I Hsp40s, T. brucei J protein 2 (Tbj2). We found that Tbj2 was heat stress-inducible, and that knockdown using RNA interference resulted in a severe growth defect under normal growth temperatures. Furthermore, a green fluorescent protein (GFP)-Tbj2 fusion protein was found to be localized to the cytosol of T. brucei. Taken together, these data suggest that Tbj2 is not functionally equivalent to the other five Type I Hsp40s, and that it is an essential, cytosolic, and stress-inducible chaperone, potentially playing an important role in protein biogenesis in T. brucei.
- Full Text:
- Date Issued: 2015
- Authors: Ludewig, Michael H , Boshoff, Aileen , Horn, David , Blatch, Gregory L
- Date: 2015
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431798 , vital:72804 , xlink:href="https://doi.org/10.1016/j.biocel.2014.12.016"
- Description: Hsp40 proteins (also known as DnaJ or J proteins) serve as co-chaperones for Hsp70, but also display evidence of independent chaperone function. Furthermore, certain Hsp40s have been shown to be stress-inducible and essential. Trypanosomatids display a remarkable diversification of Hsp40 proteins, with numerous distinct Hsp40-like proteins encoded in the Trypanosoma brucei genome. This study investigated the role of one of the six T. brucei Type I Hsp40s, T. brucei J protein 2 (Tbj2). We found that Tbj2 was heat stress-inducible, and that knockdown using RNA interference resulted in a severe growth defect under normal growth temperatures. Furthermore, a green fluorescent protein (GFP)-Tbj2 fusion protein was found to be localized to the cytosol of T. brucei. Taken together, these data suggest that Tbj2 is not functionally equivalent to the other five Type I Hsp40s, and that it is an essential, cytosolic, and stress-inducible chaperone, potentially playing an important role in protein biogenesis in T. brucei.
- Full Text:
- Date Issued: 2015
Selective modulation of plasmodial Hsp70s by small molecules with antimalarial activity
- Cockburn, Ingrid L, Boshoff, Aileen, Pesce, Eva-Rachele, Blatch, Gregory L
- Authors: Cockburn, Ingrid L , Boshoff, Aileen , Pesce, Eva-Rachele , Blatch, Gregory L
- Date: 2014
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431192 , vital:72752 , xlink:href="https://doi.org/10.1515/hsz-2014-0138"
- Description: Plasmodial heat shock protein 70 (Hsp70) chaperones represent a promising new class of antimalarial drug targets because of the important roles they play in the survival and pathogenesis of the malaria parasite Plasmodium falciparum. This study assessed a set of small molecules (lapachol, bromo-β-lapachona and malonganenones A, B and C) as potential modulators of two biologically important plasmodial Hsp70s, the parasite-resident PfHsp70-1 and the exported PfHsp70-x. Compounds of interest were assessed for modulatory effects on the steady-state basal and heat shock protein 40 (Hsp40)-stimulated ATPase activities of PfHsp70-1, PfHsp70-x and human Hsp70, as well as on the protein aggregation suppression activity of PfHsp70-x. The antimalarial marine alkaloid malonganenone A was of particular interest, as it was found to have limited cytotoxicity to mammalian cell lines and exhibited the desired properties of an effective plasmodial Hsp70 modulator. This compound was found to inhibit plasmodial and not human Hsp70 ATPase activity (Hsp40-stimulated), and hindered the aggregation suppression activity of PfHsp70-x. Furthermore, malonganenone A was shown to disrupt the interaction between PfHsp70-x and Hsp40. This is the first report to show that PfHsp70-x has chaperone activity, is stimulated by Hsp40 and can be specifically modulated by small molecule compounds.
- Full Text:
- Date Issued: 2014
- Authors: Cockburn, Ingrid L , Boshoff, Aileen , Pesce, Eva-Rachele , Blatch, Gregory L
- Date: 2014
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/431192 , vital:72752 , xlink:href="https://doi.org/10.1515/hsz-2014-0138"
- Description: Plasmodial heat shock protein 70 (Hsp70) chaperones represent a promising new class of antimalarial drug targets because of the important roles they play in the survival and pathogenesis of the malaria parasite Plasmodium falciparum. This study assessed a set of small molecules (lapachol, bromo-β-lapachona and malonganenones A, B and C) as potential modulators of two biologically important plasmodial Hsp70s, the parasite-resident PfHsp70-1 and the exported PfHsp70-x. Compounds of interest were assessed for modulatory effects on the steady-state basal and heat shock protein 40 (Hsp40)-stimulated ATPase activities of PfHsp70-1, PfHsp70-x and human Hsp70, as well as on the protein aggregation suppression activity of PfHsp70-x. The antimalarial marine alkaloid malonganenone A was of particular interest, as it was found to have limited cytotoxicity to mammalian cell lines and exhibited the desired properties of an effective plasmodial Hsp70 modulator. This compound was found to inhibit plasmodial and not human Hsp70 ATPase activity (Hsp40-stimulated), and hindered the aggregation suppression activity of PfHsp70-x. Furthermore, malonganenone A was shown to disrupt the interaction between PfHsp70-x and Hsp40. This is the first report to show that PfHsp70-x has chaperone activity, is stimulated by Hsp40 and can be specifically modulated by small molecule compounds.
- Full Text:
- Date Issued: 2014
Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK
- Nicoll, W S, Botha, M, McNamara, Caryn, Schlange, M, Pesce, E R, Boshoff, Aileen, Ludewig, M H, Zimmerman, R, Cheetham, M E, Chapple, J P, Blatch, Gregory L
- Authors: Nicoll, W S , Botha, M , McNamara, Caryn , Schlange, M , Pesce, E R , Boshoff, Aileen , Ludewig, M H , Zimmerman, R , Cheetham, M E , Chapple, J P , Blatch, Gregory L
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6484 , http://hdl.handle.net/10962/d1006261 , http://www.sciencedirect.com/science/article/pii/S1357272506003268
- Description: Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.
- Full Text:
- Date Issued: 2007
- Authors: Nicoll, W S , Botha, M , McNamara, Caryn , Schlange, M , Pesce, E R , Boshoff, Aileen , Ludewig, M H , Zimmerman, R , Cheetham, M E , Chapple, J P , Blatch, Gregory L
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6484 , http://hdl.handle.net/10962/d1006261 , http://www.sciencedirect.com/science/article/pii/S1357272506003268
- Description: Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.
- Full Text:
- Date Issued: 2007
The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum
- Shonhai, Addmore, Boshoff, Aileen, Blatch, Gregory L
- Authors: Shonhai, Addmore , Boshoff, Aileen , Blatch, Gregory L
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6486 , http://hdl.handle.net/10962/d1006269 , http://dx.doi.org/10.1110/ps.072918107
- Description: It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.
- Full Text:
- Date Issued: 2007
- Authors: Shonhai, Addmore , Boshoff, Aileen , Blatch, Gregory L
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6486 , http://hdl.handle.net/10962/d1006269 , http://dx.doi.org/10.1110/ps.072918107
- Description: It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.
- Full Text:
- Date Issued: 2007
Molecular chaperones in biology, medicine and protein biotechnology
- Boshoff, Aileen, Nicoll, William S, Hennessy, Fritha, Ludewig, M H, Daniel, Sheril, Modisakeng, Keoagile W, Shonhai, Addmore, McNamara, Caryn, Bradley, Graeme, Blatch, Gregory L
- Authors: Boshoff, Aileen , Nicoll, William S , Hennessy, Fritha , Ludewig, M H , Daniel, Sheril , Modisakeng, Keoagile W , Shonhai, Addmore , McNamara, Caryn , Bradley, Graeme , Blatch, Gregory L
- Date: 2004
- Language: English
- Type: Article
- Identifier: vital:6457 , http://hdl.handle.net/10962/d1004479
- Description: Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
- Full Text:
- Date Issued: 2004
- Authors: Boshoff, Aileen , Nicoll, William S , Hennessy, Fritha , Ludewig, M H , Daniel, Sheril , Modisakeng, Keoagile W , Shonhai, Addmore , McNamara, Caryn , Bradley, Graeme , Blatch, Gregory L
- Date: 2004
- Language: English
- Type: Article
- Identifier: vital:6457 , http://hdl.handle.net/10962/d1004479
- Description: Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
- Full Text:
- Date Issued: 2004
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