Characterization of the co-chaperones of Hsp70 and Hsp90 in Trypanosoma brucei and their potential partnerships

African Trypanosomiasis, which is caused by Trypanosoma brucei, is one of the crippling agents of social and economic development in Africa. T. brucei cycles between the cold-blooded insect vector, the tsetse fly (Glossina spp), and warm-blooded mammalian hosts. T. brucei, T. cruzi and L. major are mammal infecting kinetoplastid parasites that are collectively referred to as TriTryps. These parasites experience extreme environments as they move between their warm-blooded mammalian hosts and cold-blooded insect vectors which trigger extensive morphological transformations during the life-cycle of the parasite. Molecular chaperones have been implicated in parasite differentiation. TriTryps display significant expansions and diversity in the gene complements encoding molecular chaperones, especially J-proteins. Generally, J-proteins function as co-chaperones of Hsp70s, forming part of vital protein homeostasis processes. Hsp70s show a high degree of conservation, while J-proteins appear to be an extreme case of taxonomic radiation. Although several studies have focused on the molecular and cell biology of Hsp70s in some kinetoplastid parasites, knowledge is still lacking pertaining to J-proteins and their partnerships with Hsp70s. This thesis focused on the classification of kinetoplastid Jproteins into the four types by examining the domain organizations using T. brucei as a guide. The potential partnership of J-proteins and Hsp70s were postulated based on predicted subcellular localization. Kinetoplastid parasites, particularly T. brucei, have evolved an expanded and specialized J-protein machinery, likely to be a consequence of an evolutionary fitness/trait to adapt to diverse environment present in hosts and vectors. These analyses will yield insight into the process of parasite differentiation as well as provide new leads for chemotherapeutic treatments. The presence of the STI1 mediated Hsp90 hetero-complex formation has not been confirmed in T. brucei. To this end, in silico and biochemical techniques were used to characterize the role of TbSTI1, as an adaptor protein of Hsp70 and Hsp90. Through domain architecture analysis, sequence alignments, phylogenetic analysis and three-dimensional structure prediction, TbSTI1 was demonstrated to be the most conserved TPR containing co-chaperone of Hsp70 and Hsp83 in T. brucei and also shown to be highly similar to its eukaryotic homologues. Recombinant TbSTI1 was overproduced and purified in E.coli cells and subsequently shown to associate with TcHsp70 in a concentration dependent manner and associate weakly with TbHsp70.4. TbSTI1 and TbHsp83 were also demonstrated to be expressed and upregulated upon exposure to heat shock at the bloodstream stage of parasite development. In conclusion, this study is the first to report the interaction of TbSTI1 with a chaperone. Interactions between TbSTI1 and Hsp70s were demonstrated and therefore, the formation of the hetero-complex is predicted based the similarity of TbSTI1 to other STI1 proteins

Malarial drug targets cysteine proteases as hemoglobinases

Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor

RBBP6 interactome : RBBP6 isoform 3/DWNN and Nek6 interaction is critical for cell cycle regulation and may play a role in carcinogenesis

RBBP6 is a multidomain protein, with four splice variants translated into four functional isoforms. RBBP6 isoform 1 has been reported to interact with TP53 and pRb as well as with proteins that regulate transcriptional response to tumorigenesis such as HDM2, ZBTB38, YBX1 and NEK6. Experimental validation of isoforms 2 and 4 is yet to be conducted. The third isoform, consisting of only the DWNN domain and a short unordered c terminus, has been shown to be down-regulated in several human cancers and demonstrated as a regulator of G2/M cell cycle arrest. A number of studies have supported the role of DWNN in cell cycle regulation, however, its mechanism in these processes remains obscure. Posttranslational modification of DWNN could be critical for its function and this study was formulated to understand how the DWNN regulates the cell cycle. Our study identified 12 cell cycle-related proteins interacting with DWNN using various bioinformatics tools. We also identified 10 ubiquitin ligases that interact with DWNN. The most relevant interacting partner, the cell cycle regulator Nek6, has been reported to interact with DWNN during the cell cycle. It was therefore critical to interrogate the interaction between Nek6 and DWNN by homology modelling and docking. The DWNN mutants had a reduced affinity for NEK6 with at least one of the mutants having changes that affect at least one phosphorylation site. It is likely that NEK6 promotes cell proliferation by phosphorylating DWNN. This work suggests that DWNN co-regulates RNA splicing, ubiquitination, and cell cycle control. DWNN may therefore, be targeted for novel anticancer therapies through cell cycle regulation.Supplementary Material: Fig. S1. Comparison of the Ramachandran plots for Nek 7 as determined by crystallography and the Nek6 models using Nek 7 as a templateFig. S2. Structure of wild-type RBBP5 isoform 3 compared to mutant forms of the protein: (A) The wild type RBBP6 isoform 3 structure as determined by NMR. The protein has 7 Beta sheets and one alpha helix. (B) A 3D model of the S25A mutant of RBBP6 isoform 3 and (C) the 3D model of the T49A mutant of RBBP6 isoform 3, show that the amino acid substitutions in the mutant proteins had no significant effect on the overall structure of the protein. Like the wild type protein, the mutants all contain 7 Beta sheets and one alpha helixThe South African Medical Research Council (SAMRC) and the National Research Foundation (NRF).https://http//www.elsevier.com/locate/imuhj2021Obstetrics and Gynaecolog

Stress biology:Complexity and multifariousness in health and disease

Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.</p

Pharmacophore Model-Based Virtual Screening Workflow for Discovery of Inhibitors Targeting <i>Plasmodium falciparum</i> Hsp90

Plasmodium falciparum causes the most lethal and widespread form of malaria. Eradication of malaria remains a priority due to the increasing number of cases of drug resistance. The heat shock protein 90 of P. falciparum (PfHsp90) is a validated drug target essential for parasite survival. Most PfHsp90 inhibitors bind at the ATP binding pocket found in its N-terminal domain, abolishing the chaperone's activities, which leads to parasite death. The challenge is that the NTD of PfHsp90 is highly conserved, and its disruption requires selective inhibitors that can act without causing off-target human Hsp90 activities. We endeavored to discover selective inhibitors of PfHsp90 using pharmacophore modeling, virtual screening protocols, induced fit docking (IFD), and cell-based and biochemical assays. The pharmacophore model (DHHRR), composed of one hydrogen bond donor, two hydrophobic groups, and two aromatic rings, was used to mine commercial databases for initial hits, which were rescored to 20 potential hits using IFD. Eight of these compounds displayed moderate to high activity toward P. falciparum NF54 (i.e., IC50s ranging from 6.0 to 0.14 μM) and averaged >10 in terms of selectivity indices toward CHO and HepG2 cells. Additionally, four compounds inhibited PfHsp90 with greater selectivity than a known inhibitor, harmine, and bound to PfHsp90 with weak to moderate affinity. Our findings support the use of a pharmacophore model to discover diverse chemical scaffolds such as FM2, FM6, F10, and F11 exhibiting anti-Plasmodium activities and serving as valuable new PfHsp90 inhibitors. Optimization of these hits may enable their development into potent leads for future antimalarial drugs