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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.

Co-chaperonins function together with chaperonins to mediate ATP-dependent protein folding in a variety of cellular compartments. Chaperonins are evolutionarily conserved and form two distinct classes, namely, group I and group II chaperonins. GroEL and its co-chaperonin GroES form part of group I and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo conformational rearrangements that enable protein folding to occur. GroES forms a lid over the chamber and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. Group II chaperonins are functionally similar to group I chaperonins but differ in structure and do not require a co-chaperonin. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances, co-chaperonins display contrasting functions to those of chaperonins. Human HSP60 (HSPD) continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10 on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

African trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei (T. brucei) and spread by the tsetse fly in sub-Saharan Africa. The trypanosome relies on heat shock proteins for survival in the insect vector and mammalian host. Heat shock protein 90 (HSP90) plays a crucial role in the stress response at the cellular level. Inhibition of its interactions with chaperones and co-chaperones is being explored as a potential therapeutic target for numerous diseases. This study provides an in silico overview of HSP90 and its co-chaperones in both T. brucei brucei and T. brucei gambiense in relation to human and other trypanosomal species, including non-parasitic Bodo saltans and the insect infecting Crithidia fasciculata. A structural analysis of T. brucei HSP90 revealed differences in the orientation of the linker and C-terminal domain in comparison to human HSP90. Phylogenetic analysis displayed the T. brucei HSP90 proteins clustering into three distinct groups based on subcellular localizations, namely, cytosol, mitochondria, and endoplasmic reticulum. Syntenic analysis of cytosolic HSP90 genes revealed that T. b. brucei encoded for 10 tandem copies, while T. b. gambiense encoded for three tandem copies; Leishmania major (L. major) had the highest gene copy number with 17 tandem copies. The updated information on HSP90 from recently published proteomics on T. brucei was examined for different life cycle stages and subcellular localizations. The results show a difference between T. b. brucei and T. b. gambiense with T. b. brucei encoding a total of twelve putative HSP90 genes, while T. b. gambiense encodes five HSP90 genes. Eighteen putative co-chaperones were identified with one notable absence being cell division cycle 37 (Cdc37). These results provide an updated framework on approaching HSP90 and its interactions as drug targets in the African trypanosome.

Molecular chaperones are a group of structurally diverse and highly conserved ubiquitous proteins. They play crucial roles in facilitating the correct folding of proteins in vivo by preventing protein aggregation or facilitating the appropriate folding and assembly of proteins. Heat shock proteins form the major class of molecular chaperones that are responsible for protein folding events in the cell. This is achieved by ATP-dependent (folding machines) or ATP-independent mechanisms (holders). Heat shock proteins are induced by a variety of stresses, besides heat shock. The large and varied heat shock protein class is categorised into several subfamilies based on their sizes in kDa namely, small Hsps (HSPB), J domain proteins (Hsp40/DNAJ), Hsp60 (HSPD/E; Chaperonins), Hsp70 (HSPA), Hsp90 (HSPC), and Hsp100. Heat shock proteins are localised to different compartments in the cell to carry out tasks specific to their environment. Most heat shock proteins form large oligomeric structures, and their functions are usually regulated by a variety of cochaperones and cofactors. Heat shock proteins do not function in isolation but are rather part of the chaperone network in the cell. The general structural and functional features of the major heat shock protein families are discussed, including their roles in human disease. Their function is particularly important in disease due to increased stress in the cell. Vector-borne parasites affecting human health encounter stress during transmission between invertebrate vectors and mammalian hosts. Members of the main classes of heat shock proteins are all represented in Plasmodium falciparum, the causative agent of cerebral malaria, and they play specific functions in differentiation, cytoprotection, signal transduction, and virulence.

Trypanosoma brucei (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against T. brucei.

African Trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei (T. brucei) and is spread by the tsetse fly in sub-Saharan Africa. The disease is fatal if left untreated and the currently approved drugs for treatment are toxic and difficult to administer. The trypanosome must survive in the insect vector and its mammalian host, and to adapt to these different conditions, the parasite relies on molecular chaperones called heat shock proteins. Heat shock proteins mediate the folding of newly synthesized proteins as well as prevent misfolding of proteins under normal conditions and during stressful conditions. Heat shock protein 90 (Hsp90) is one of the major molecular chaperones of the stress response at the cellular level. It functions with other chaperones and co-chaperones and inhibition of its interactions is being explored as a potential therapeutic target for numerous diseases. This study provides an in-silico overview of Hsp90 and its co-chaperones in both T. brucei brucei and T. brucei gambiense in relation to human and other kinetoplastid parasites. The evolutionary, functional, and structural analyses of Hsp90 were also shown. The updated information on Hsp90 and its co-chaperones from recently published proteomics on T. brucei was examined for the different life cycle stages and subcellular localisations. The results show a difference between T. b. brucei and T. b. gambiense with T. b. brucei encoding 12 putative Hsp90 genes, 10 of which are cytosolic and located on a single chromosome while T. gambiense encodes 5 Hsp90 genes, 3 of which are located in the cytosol. Eight putative co-chaperones were identified in this study, 6 TPR-containing and 2 non-TPR-containing co-chaperones. This study provides an updated context for studying the biology of the African trypanosome and evaluating Hsp90 and its interactions as potential drug targets.

The etiological agent of the neglected tropical disease African trypanosomiasis, Trypanosoma brucei, possesses an expanded and diverse repertoire of heat shock proteins, which have been implicated in cytoprotection, differentiation, as well as progression and transmission of the disease. Hsp70 plays a crucial role in proteostasis, and inhibition of its interactions with co-chaperones is emerging as a potential therapeutic target for numerous diseases. In light of genome annotations and the release of the genome sequence of the human infective subspecies, an updated and current in silico overview of the Hsp70/J-protein machinery in both T. brucei brucei and T. brucei gambiense was conducted. Functional, structural, and evolutionary analyses of the T. brucei Hsp70 and J-protein families were performed. The Hsp70 and J-proteins from humans and selected kinetoplastid parasites were used to assist in identifying proteins from T. brucei, as well as the prediction of potential Hsp70–J-protein partnerships. The Hsp70 and J-proteins were mined from numerous genome-wide proteomics studies, which included different lifecycle stages and subcellular localisations. In this study, 12 putative Hsp70 proteins and 67 putative J-proteins were identified to be encoded on the genomes of both T. brucei subspecies. Interestingly there are 6 type III J-proteins that possess tetratricopeptide repeat-containing (TPR) motifs. Overall, it is envisioned that the results of this study will provide a future context for studying the biology of the African trypanosome and evaluating Hsp70 and J-protein interactions as potential drug targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-018-0950-x) contains supplementary material, which is available to authorized users.

Hsp70 members occupy a central role in proteostasis and are found in different eukaryotic cellular compartments. The mitochondrial Hsp70/J-protein machinery performs multiple functions vital for the proper functioning of the mitochondria, including forming part of the import motor that transports proteins from the cytosol into the matrix and inner membrane, and subsequently folds these proteins in the mitochondria. However, unlike other Hsp70s, mitochondrial Hsp70 (mtHsp70) has the propensity to self-aggregate, accumulating as insoluble aggregates. The self-aggregation of mtHsp70 is caused by both interdomain and intramolecular communication within the ATPase and linker domains. Since mtHsp70 is unable to fold itself into an active conformation, it requires an Hsp70 escort protein (Hep) to both inhibit self-aggregation and promote the correct folding. Hep1 orthologues are present in the mitochondria of many eukaryotic cells but are absent in prokaryotes. Hep1 proteins are relatively small and contain a highly conserved zinc-finger domain with one tetracysteine motif that is essential for binding zinc ions and maintaining the function and solubility of the protein. The zinc-finger domain lies towards the C-terminus of Hep1 proteins, with very little conservation outside of this domain. Other than maintaining mtHsp70 in a functional state, Hep1 proteins play a variety of other roles in the cell and have been proposed to function as both chaperones and co-chaperones. The cellular localisation and some of the functions are often speculative and are not common to all Hep1 proteins analysed to date.

Introduction: South Africa is a member state of the “BRICS” bloc (BRICS2017.org, 2017) and the G20 group of the 20 nations/economic blocs, which between them account for the majority of the world’s trade and economic activity. It faces many developmental challenges which are mirrored in its higher education sector. In this article, the authors seek to provide an overview of the challenges that South African higher education faces in the achievement of the developmental goals of the country. The focus of this paper is a case study in WASH (water, sanitation and hygiene) to improve context-specific responses that trains pharmacists on knowledge and skills. Methods: The study was performed as a combination of calculations and a literature review to obtain the background or current status of the higher education sector and developmental planning in South Africa. For this, data were extracted from the Statistics South Africa reports, relevant professional articles on South African higher education sector and results of postgraduate research. Workshop results which were obtained as a collaboration between a public and a private higher education institution and results of postgraduate research were used as the paradigm for transformation and decolonisation of the curriculum for a professional degree in South Africa. Results and discussion: Challenges exist in the South African tertiary education sector and the graduation rate currently stands at 65.1% of the target set by the National Development Plan. Around 58.1% of all students do not complete their university/post-secondary education, which could provide a partial explanation for the skills shortage in South Africa. Decolonisation and transformation of the tertiary education curriculum are major topics in the discourse on higher education in South Africa. The authors propose that one way to achieve this would be inclusion of research results and group activities in the area of water, sanitation and hygiene as a topic for possible and partial transformation of the Bachelor of Pharmacy curriculum. Conclusions: The current article summarises some of topics and challenges that drive the current discourse, developmental and curriculum debate in higher education in South Africa. Student access and through put at tertiary institutions need to be improved and the curriculum needs to be transformed.

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.

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.

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.

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.

Many biological processes in the cell are linked to RNA metabolism and therefore have implications for a wide range of biotechnological applications. The processing and degradation of RNA plays an important role in RNA metabolism with often the same enzymes being involved in both processes. In this review, we highlight the dynamic nature of the structural components of the Escherichia coli RNA degradosome which is a large multiprotein complex that plays an important role in RNA degradation. The activities of the individual components of the degradosome are also discussed. The RNA degradosome forms part of the bacterial cytoskeleton and associated proteins, such as molecular chaperones, may aid in the compartmentalization of enzymatic activities and cytoskeletal organization. An enhanced understanding of the components of the RNA degradosome in other bacterial species will certainly aid in their evaluation as potential antimicrobial agents.

The picornavirus 2C protein is highly conserved and indispensible for virus replication. Polyclonal antibodies against Theiler's murine encephalomyelitis virus (TMEV) 2C protein were generated by immunisation of rabbits with a peptide comprising amino acids 31-210 of the protein. Antibodies were used to investigate the localisation of 2C in infected cells by indirect immunofluorescence and confocal microscopy. Analysis of infected cells revealed that the distribution of 2C changed during infection. Early on, the protein was localised in the perinuclear region with punctate staining in the cytoplasm and at later stages, it was concentrated in one large structure in close proximity to the nucleus and occupying almost 50% of the cell size. Dual-label immunofluorescence using wheat germ agglutinin (WGA) and anti-TMEV 2C antibodies suggested that 2C, and therefore virus replication, is targeted to the Golgi apparatus. At late stages of infection Golgi staining was dispersed, indicating potential reorganisation of membranes. Infection was accompanied by "rounding up" of the cells and a redistribution of actin around the putative replication complex. The results suggest that TMEV behaves similarly to FMDV which also forms replication complexes in the perinuclear region.

DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.

Co-chaperonins function together with chaperonins to mediate ATP-dependant protein folding in a variety of cellular compartments. GroEL and its co-chaperonin GroES are the only essential chaperones in Escherichia coli and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo structural rearrangements as part of the folding mechanism. GroES forms a lid over the chamber, and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances co-chaperonins display contrasting functions to those of chaperonins. Human Hsp60 continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10, in addition to its role as a co-chaperonin, on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

Co-chaperonins function together with chaperonins to mediate ATP-dependant protein folding in a variety of cellular compartments. GroEL and its co-chaperonin GroES are the only essential chaperones in Escherichia coli and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo structural rearrangements as part of the folding mechanism. GroES forms a lid over the chamber, and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances co-chaperonins display contrasting functions to those of chaperonins. Human Hsp60 continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10, in addition to its role as a co-chaperonin, on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

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.

Polyphenol oxidase (PPO), obtained from Agaricus bisporus, can be used in hydroxylating a range of phenolic substrates to yield catechols which are then oxidised by the enzyme to give o-quinone products. The objective of this study was to develop systems whereby phenols could be transformed by PPO, and the products of the biotransformation could be isolated and characterised. By comparing the product mixtures obtained using soluble PPO and various forms of immobilised PPO, in aqueous and non-aqueous media, we have found significant differences in reaction rates and in the proportions of catechol and quinone produced. PPO in solution is inactivated by the reaction products, but when it is immobilised, the separation of products from the enzyme reduces this inhibition. Immobilisation also leads to increased stability, and allows continuous use of the enzyme. In bioreactors containing customised novel asymmetric capillary membranes as the enzyme support, high concentrations of phenolic substrates were converted. The addition of a chitosan-containing column downstream from the capillary membrane bioreactor facilitated the removal of the coloured quinone products from the permeate, and recycling of the substrate solution.

Preliminary studies were made of the potential use of a membrane-immobilised enzymatic method for the treatment of p-cresol-containing wastewater. The enzyme polyphenol oxidase was immobilised onto hydrophobic polyethersulphone capillary membranes and hydrophilic nylon flat-sheet membranes, by adsorption, and by adsorption with glutaraldehyde cross-linking, respectively. It was found that the intermediate product of the polyphenol oxidase reaction, 4-methylcatechol, was detected when the enzyme was immobilised on the nylon membranes or was not immobilised, but only the o-quinone final product was detected when polyethersulphone was used as the immobilisation matrix.

Molecular chaperones are a group of structurally diverse and highly conserved ubiquitous proteins. They play crucial roles in facilitating the correct folding of proteins in vivo by preventing protein aggregation or facilitating the appropriate folding and assembly of proteins. Heat shock proteins form the major class of molecular chaperones that are responsible for protein folding events in the cell. This is achieved by ATP-dependent (folding machines) or ATP-independent mechanisms (holders). Heat shock proteins are induced by a variety of stresses, besides heat shock. The large and varied heat shock protein class is categorised into several subfamilies based on their sizes in kDa namely, small Hsps (HSPB), Hsp40 (DNAJ), Hsp60 (HSPD/E; Chaperonins), Hsp70 (HSPA), Hsp90 (HSPC), and Hsp100. Heat shock proteins are localised to different compartments in the cell to carry out tasks specific to their environment. Most heat shock proteins form large oligomeric structures and their functions are usually regulated by a variety of cochaperones and cofactors. Heat shock proteins do not function in isolation but are rather part of the chaperone network in the cell. The general structural and functional features of the major heat shock protein families are discussed, including their roles in human disease. Their function is particularly important in disease due to increased stress in the cell. Vector-borne parasites affecting human health encounter stress during transmission between invertebrate vectors and mammalian hosts. Members of the main classes of heat shock proteins are all represented in Plasmodium falciparum, the causative agent of cerebral malaria, and they play specific functions in differentiation, cytoprotection, signal transduction and virulence.

Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) is a key enzyme in the synthesis of isoprenoids in the malaria parasite, using a pathway that is absent in the human host. This enzyme is receiving attention as it has been validated as a promising drug target. However, an impediment to the characterisation of this en- zyme has been the inability to obtain sufficient quantities of the enzyme in a soluble and functional form. The expression of PfDXR from the codon harmonised coding region, under conditions of strongly controlled transcription and induction, resulted in a yield of 2 - 4 mg/L of enzyme, which is 8 to 10-fold higher than previously reported yields. The kinetic pa- rameters Km, Vmax and kcat were determined for PfDXR using an NADPH-dependent assay. Residues K295 and K297, unique to species of Plasmodium and located in the catalytic hatch region; and residues V114 and N115, essential for NADPH binding, were mutated to resemble those found in E. coli DXR. Interestingly, these mutations decreased the sub- strate affinity of PfDXR to values resembling that of E. coli DXR. PfDXR-K295N, K297S and PfDXR-V114A, N115G demonstrated a decreased ability to turnover substrate by 4-fold and 2-fold respectively in comparison to PfDXR. This study indicates a difference in the role of the catalytic hatch in capturing substrate by species of Plasmodium. The results of this study could contribute to the development of inhibitors of PfDXR.

The chaperonin protein GroEL was mixed with varying concentrations of K2PtCl4 followed by a 20-fold concentration of sodium borohydride to afford GroEL–platinum nanoparticle complexes in a ratio of between 1:25 and 1:2,000. Typical colour change, from colourless or pale yellow to brown, occurred that was dependent on the amount of platinum present. These complexes were characterised by UV/Vis, inductively coupled plasma optical emission spectroscopy, Fourier transform infra red, transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy. TEM analysis revealed that the size of nanoparticles increased as the molar ratio of platinum to GroEL increased with an average size diameter of 1.72–3.5 nm generated with GroEL–platinum molar ratios of 1:125–1:2,000. Fourier-transform infrared spectroscopy (FTIR) spectra showed no distinct changes in the structure of GroEL but confirmed that the nanoparticles were attached to the protein. The effect of platinum nanoparticles on the ATPase activity of GroEL showed an activity of 5.60 μmol min−1 ml−1 (87 % increase over a control) at the molar ratio of GroEL–platinum nanoparticles of 1:25.

Molecular chaperones are integral components of the cellular machinery involved in ensuring correct protein folding and the continued maintenance of protein structure. An understanding of these ubiquitous molecules is key to finding cures to protein misfolding diseases such as Alzheimer's and Creutzfeldt-Jacob diseases. In addition, further understanding of chaperones will enhance our comprehension of the way the body copes with the environmental stresses that humans encounter daily. Our laboratory and our collaborators specialize in the production and characterization of chaperones from a wide variety of sources in order to gain a fuller understanding of how chaperones function in the cell. In this review, we primarily use the Hsp70/Hsp40 chaperone pair as an example to discuss recent advances in technology and reductions in cost that lend themselves to chaperone purification from both native and recombinant sources. Common assays to assess purified chaperone activity are also discussed.

Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp 70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp 70 was able to suppress the thermosensitivity of an Escherichia coli dnaK 756 strain, but not that of the corresponding deletion strain (DeltadnaK 52) or dnaK 103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp 70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp 70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK 756 strain. We also identified two key amino acid residues (V 401 and Q 402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp 70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp 70 and PfHsp 70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp 40 chaperone machinery.

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 microM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.

Prokaryotic DnaJ and DnaK, homologous to the eukaryotic 40 and 70kDa heat shock proteins (Hsp40 and Hsp70) respectively, play an important role as molecular chaperones in assisted protein folding under both normal and stressed conditions. DnaJ-like proteins are defined by the presence of a 70 amino acid domain termed the J domain, similar to the initial 73 amino acids of the Escherichia coli protein DnaJ. The J domain comprises four alpha-helices and a loop region containing the invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif and Helix II have been shown previously to be important for the interaction with partner Hsp70s. Conserved amino acid residues present in the J domain were identified, and substitutions of these residues were performed to examine their effect on the in vivo functioning of the J domain of Agrobacterium tumefaciens DnaJ. Three conserved, charged residues, and three conserved, hydrophobic residues, in addition to the HPD motif, were shown to be important for the correct functioning of A. tumefaciens DnaJ. These included Arg26 located on Helix II, Arg63 and Asp59 located on Helix IV, Tyr7 and Leu10 located on Helix I, and Leu57 located on Helix III. This study has identified charged and hydrophobic residues on all the structural elements of the J domain that were critical to the structure and function of DnaJ, and in particular shown that Helix IV may have an important role in the structure and function of DnaJs in general.

Plasmodium falciparum heat shock protein (PfHsp70) has been proposed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. However, the biochemical and chaperone properties of PfHsp70 have not been elucidated. The heterologous overproduction of P. falciparum proteins in Escherichia coli is problematic because of its AT-rich genome and the usage of codons that are rarely used in E. coli. In this paper, we describe the successful overproduction of (His)(6)-PfHsp70 in E. coli using the pQE30 expression vector system. Initial experiments with E. coli [pQE30/PfHsp70] resulted in the overproduction of the full-length protein and truncated derivatives. The RIG plasmid, which encodes tRNAs for rare codons, was engineered into the E. coli [pQE30/PfHsp70] strain, resulting in significant reduction of the truncated (His)(6)-PfHsp70 derivatives and improved yields of the full-length protein. (His)(6)-PfHsp70 was successfully purified using nickel-chelating Sepharose affinity chromatography and its biochemical properties were determined. The V(max), K(m), and k(cat) for the basal ATPase activity of (His)(6)-PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 microM, and 1.03 min(-1), respectively. Gel filtration studies indicated that (His)(6)-PfHsp70 existed largely as a monomer in solution. This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.

Although previous research has focused on phenol removal efficiencies using polyphenol oxidase in nonimmobilized and immobilized forms, there has been little consideration of the use of polyphenol oxidase in a biotransformation system for the production of catechols. In this study, polyphenol oxidase was successfully immobilized on various synthetic membranes and used to convert phenolic substrates to catechol products. A neural network model was developed and used to model the rates of substrate utilization and catechol production for both nonimmobilized and immobilized polyphenol oxidase. The results indicate that the biotransformation of the phenols to their corresponding catechols was strongly influenced by the immobilization support, resulting in differing yields of catechols. Hydrophilic membranes were found to be the most suitable immobilization supports for catechol production. The successful biocatalytic production of 3-methylcatechol, 4-methylcatechol, catechol, and 4-chlorocatechol is demonstrated.