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.

Ursolic acid acetate and iso-mukaadial acetate bind to Plasmodium falciparum Hsp90, abrogating its chaperone function in vitro.

Plasmodium falciparum is the most lethal malaria parasite. Increasing incidences of drug resistance of P. falciparum have prompted the need for discovering new and effective antimalarial compounds with an alternative mode of action. Heat shock protein 90 (PfHsp90) facilitates protein folding and is a promising antimalarial drug target. We have previously reported that iso-mukaadial acetate (IMA) and ursolic acid acetate (UAA) exhibit antimalarial activity. We investigated the abilities of IMA and UAA to bind PfHsp90 by molecular docking and dynamics simulations. The in silico predictions were validated by biochemical assays conducted on recombinant PfHsp90. The interaction between the ligands and PfHsp90 was evaluated using ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared (FTIR), and surface plasmon resonance (SPR) analysis. The results obtained by docking calculations and MD dynamics simulation predicted that UAA and IMA preferentially bound to PfHsp90 via the N-terminal domain, with UAA binding more stable than IMA. UV-vis-based data suggest that PfHsp90 harbors buried aromatic amino acids, which were exposed in the presence of either IMA or UAA. In addition, data obtained using FTIR suggested that IMA and UAA destabilized the secondary structure of PfHsp90. Of the two compounds, UAA bound to PfHsp90 within the micromolar range based on surface plasmon resonance (SPR)-based binding assay. Furthermore, both compounds disrupted the holdase chaperone function of PfHsp90 as the chaperone failed to suppress heat-induced aggregation of the model proteins, malate dehydrogenase (MDH), luciferase, and citrate synthase in vitro. In addition, both compounds lowered the ATPase activity of PfHsp90. The molecular dynamics simulation analysis indicated that the docked complexes were mostly stable for 100 ns, validating the data obtained through the biochemical assays. Altogether, this study expands the repository of antiplasmodial compounds that have PfHsp90 among their possible targets.

Insertion of GGMP repeat residues of Plasmodium falciparum Hsp70-1 in the lid of DnaK adversely impacts client recognition.

Although Hsp70 is a conserved molecular chaperone, it exhibits some degree of functional specialisation across species. Features of Hsp70 regulating its functional specialisation remain to be fully established. We previously demonstrated that E. coli Hsp70 (DnaK) exhibits functional features that distinguishes it from PfHsp70-1, a canonical cytosolic Hsp70 of Plasmodium falciparum. One of the defining features of PfHsp70-1 is that it possesses GGMP repeat residues located in its C-terminal lid segment, while DnaK lacks this motif. Previously, we demonstrated that the insertion of GGMP repeat residues of PfHsp70-1 into E. coli DnaK abrogates the chaperone activity of DnaK. However, the role of the GGMP motif in regulating Hsp70 function remains to be fully understood. To explore the function of this motif, we expressed recombinant forms of wild type DnaK and its GGMP insertion motif, DnaK-G and systematically characterised the structure-function features of the two proteins using in silico analysis, biophysical approaches and an in cellulo complementation assay. Our findings demonstrated that the GGMP inserted in DnaK compromised various functional features such as nucleotide binding, allostery, substrate binding affinity and cellular proteome client selectivity. These findings thus, highlight the GGMP motif of Hsp70 as an important functional module.

Comparative genomics and proteomics analysis of phages infecting multi-drug resistant Escherichia coli O177 isolated from cattle faeces.

The increasing prevalence of antimicrobial-resistant (AMR) pathogens has become a major global health concern. To address this challenge, innovative strategies such as bacteriophage therapy must be optimised. Genomic characterisation is a crucial step in identifying suitable phage candidates for combating AMR pathogens. The aim of this study was to characterise seven phages that infect the Escherichia coli O177 strain using a whole genome sequencing. The analysis of genome sequences revealed that these phages had linear dsDNA, with genome sizes spanning from 136, 483 to 166,791 bp and GC content varying from 35.39 to 43.63%. Taxonomically, the phages were classified under three different subfamilies (Stephanstirmvirinae, Tevenvirinae, and Vequintavirinae) and three genera (Phapecoctavirus, Tequatrovirus, and Vequintavirus) within the class Caudoviricetes. In silico PhageAI analysis predicted that all the phages were virulent, with confidence levels between 96.07 and 97.26%. The phage genomes contained between 66 and 82 ORFs, which encode hypothetical and putative functional proteins. In addition, the phage genomes contained core genes associated with molecular processes such as DNA replication, transcription modulation, nucleotide metabolism, phage structure (capsid and tail), and lysis. None of the genomes carried genes associated with undesirable traits such as integrase, antimicrobial resistance, virulence, and toxins. The study revealed high genome and proteome homology among E. coli O177 phages and other known Escherichia phages. The results suggest that the seven phages are new members of the genera Phapecoctavirus, Tequatrovirus, and Vequintavirus under the subfamilies Stephanstirmvirinae, Tevenvirinae, and Vequintavirinae, respectively.

Pharmacophore Model-Based Virtual Screening Workflow for Discovery of Inhibitors Targeting Plasmodium falciparum 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.

Whole Genome Analysis of African G12P[6] and G12P[8] Rotaviruses Provides Evidence of Porcine-Human Reassortment at NSP2, NSP3, and NSP4.

Group A rotaviruses (RVA) represent the most common cause of pediatric gastroenteritis in children <5 years, worldwide. There has been an increase in global detection and reported cases of acute gastroenteritis caused by RVA genotype G12 strains, particularly in Africa. This study sought to characterize the genomic relationship between African G12 strains and determine the possible origin of these strains. Whole genome sequencing of 34 RVA G12P[6] and G12P[8] strains detected from the continent including southern (South Africa, Zambia, Zimbabwe), eastern (Ethiopia, Uganda), central (Cameroon), and western (Togo) African regions, were sequenced using the Ion Torrent PGM method. The majority of the strains possessed a Wa-like backbone with consensus genotype constellation of G12-P[6]/P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while a single strain from Ethiopia displayed a DS-1-like genetic constellation of G12-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. In addition, three Ethiopian and one South African strains exhibited a genotype 2 reassortment of the NSP3 gene, with genetic constellation of G12-P[8]-I1-R1-C1-M1-A1-N1-T2-E1-H1. Overall, 10 gene segments (VP1-VP4, VP6, and NSP1-NSP5) of African G12 strains were determined to be genetically related to cognate gene sequences from globally circulating human Wa-like G12, G9, and G1 strains with nucleotide (amino acid) identities in the range of 94.1-99.9% (96.5-100%), 88.5-98.5% (93-99.1%), and 89.8-99.0% (88.7-100%), respectively. Phylogenetic analysis showed that the Ethiopian G12P[6] possessing a DS-1-like backbone consistently clustered with G2P[4] strains from Senegal and G3P[6] from Ethiopia with the VP1, VP2, VP6, and NSP1-NSP4 genes. Notably, the NSP2, NSP3, and NSP4 of most of the study strains exhibited the closest relationship with porcine strains suggesting the occurrence of reassortment between human and porcine strains. Our results add to the understanding of potential roles that interspecies transmission play in generating human rotavirus diversity through reassortment events and provide insights into the evolutionary dynamics of G12 strains spreading across selected sub-Saharan Africa regions.

Abnormalities in alternative splicing in diabetes: therapeutic targets.

Diabetes mellitus (DM) is a non-communicable, metabolic disorder that affects 416 million individuals worldwide. Type 2 diabetes contributes to a vast 85-90% of the diabetes incidences while 10-15% of patients suffer from type 1 diabetes. These two predominant forms of DM cause a significant loss of functional pancreatic ß-cell mass causing different degrees of insulin deficiency, most likely, due to increased ß-cell apoptosis. Treatment options involve the use of insulin sensitisers, α-glucosidase inhibitors, and ß-cell secretagogues which are often expensive, limited in efficacy and carry detrimental adverse effects. Cost-effective options for treatment exists in the form of herbal drugs, however, scientific validations of these widely used medicinal plants are still underway. Alternative splicing (AS) is a co-ordinated post-transcriptional process in which a single gene generates multiple mRNA transcripts which results in increased amounts of functionally different protein isoforms and in some cases aberrant splicing leads to metabolic disease. In this review, we explore the association of AS with metabolic alterations in DM and the biological significance of the abnormal splicing of some pathogenic diabetes-related genes. An understanding of the molecular mechanism behind abnormally spliced transcripts will aid in the development of new diagnostic, prognostic and therapeutic tools.