In silico analysis of the HSP90 chaperone system from the African trypanosome, Trypanosoma brucei.

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.

Characterization of an Atypical Trypanosoma brucei Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue.

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.

Trypanosoma brucei J-Protein 2 Functionally Co-Operates with the Cytosolic Hsp70 and Hsp70.4 Proteins.

The etiological agent of African trypanosomiasis, Trypanosoma brucei (Tb), has been identified to possess an expanded and diverse group of heat shock proteins, which have been implicated in cytoprotection, differentiation, and subsequently progression and transmission of the disease. Heat shock protein 70 (Hsp70) is a highly conserved and ubiquitous molecular chaperone that is important in maintaining protein homeostasis in the cell. Its function is regulated by a wide range of co-chaperones, and inhibition of these functions and interactions with co-chaperones are emerging as potential therapeutic targets for numerous diseases. This study sought to biochemically characterize the cytosolic TbHsp70 and TbHsp70.4 proteins and to investigate if they functionally co-operate with the Type I J-protein, Tbj2. Expression of TbHsp70 was shown to be heat inducible, while TbHsp70.4 was constitutively expressed. The basal ATPase activities of TbHsp70.4 and TbHsp70 were stimulated by Tbj2. It was further determined that Tbj2 functionally co-operated with TbHsp70 and TbHsp70.4 as the J-protein was shown to stimulate the ability of both proteins to mediate the refolding of chemically denatured ß-galactosidase. This study provides further insight into this important class of proteins, which may contribute to the development of new therapeutic strategies to combat African Trypanosomiasis.

The Hsp70/J-protein machinery of the African trypanosome, Trypanosoma brucei.

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.