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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.

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.

Tsetse flies (Glossina spp.) are the sole vectors of the protozoan parasites of the genus Trypanosoma, the causative agents of African Trypanosomiasis. Species of Glossina differ in vector competence and Glossina morsitans morsitans is associated with transmission of Trypanosoma brucei rhodesiense, which causes an acute and often fatal form of African Trypanosomiasis. Heat shock proteins are evolutionarily conserved proteins that play critical roles in proteostasis. The activity of heat shock protein 70 (Hsp70) is regulated by interactions with its J-protein (Hsp40) co-chaperones. Inhibition of these interactions are emerging as potential therapeutic targets. The assembly and annotation of the G. m. morsitans genome provided a platform to identify and characterize the Hsp70s and J-proteins, and carry out an evolutionary comparison to its well-studied eukaryotic counterparts, Drosophila melanogaster and Homo sapiens, as well as Stomoxys calcitrans, a comparator species. In our study, we identified 9 putative Hsp70 proteins and 37 putative J-proteins in G. m. morsitans. Phylogenetic analyses revealed three evolutionarily distinct groups of Hsp70s, with a closer relationship to orthologues from its blood-feeding dipteran relative Stomoxys calcitrans. G. m. morsitans also lacked the high number of heat inducible Hsp70s found in D. melanogaster. The potential localisations, functions, domain organisations and Hsp70/J-protein partnerships were also identified. A greater understanding of the heat shock 70 (Hsp70) and J-protein (Hsp40) families in G. m. morsitans could enhance our understanding of the cell biology of the tsetse fly.

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.

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.

The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

Abstract Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) 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 (PfHsp70) 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 PfHsp70 was able to suppress the thermosensitivity of an Escherichia coli dnaK756 strain, but not that of the corresponding deletion strain (ΔdnaK52) or dnaK103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp70 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 PfHsp70 (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 dnaK756 strain. We also identified two key amino acid residues (V401 and Q402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp70. 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 PfHsp70 and PfHsp70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp40 chaperone machinery. [ABSTRACT FROM AUTHOR]