A native mass spectrometry approach to qualitatively elucidate interfacial epitopes of transient protein-protein interactions.

Native mass spectrometric analysis of TPR2A and GrpE with unpurified peptides derived from limited proteolysis of their respective PPI partners (HSP90 C-terminus and DnaK) facilitated efficient, qualitative identification of interfacial epitopes involved in transient PPI formation. Application of this approach can assist in elucidating interfaces of currently uncharacterised transient PPIs.

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.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

Tackling Sleeping Sickness: Current and Promising Therapeutics and Treatment Strategies.

Human African trypanosomiasis is a neglected tropical disease caused by the extracellular protozoan parasite Trypanosoma brucei, and targeted for eradication by 2030. The COVID-19 pandemic contributed to the lengthening of the proposed time frame for eliminating human African trypanosomiasis as control programs were interrupted. Armed with extensive antigenic variation and the depletion of the B cell population during an infectious cycle, attempts to develop a vaccine have remained unachievable. With the absence of a vaccine, control of the disease has relied heavily on intensive screening measures and the use of drugs. The chemotherapeutics previously available for disease management were plagued by issues such as toxicity, resistance, and difficulty in administration. The approval of the latest and first oral drug, fexinidazole, is a major chemotherapeutic achievement for the treatment of human African trypanosomiasis in the past few decades. Timely and accurate diagnosis is essential for effective treatment, while poor compliance and resistance remain outstanding challenges. Drug discovery is on-going, and herein we review the recent advances in anti-trypanosomal drug discovery, including novel potential drug targets. The numerous challenges associated with disease eradication will also be addressed.

Bimolecular Fluorescence Complementation Assay to Evaluate HSP90-Client Protein Interactions in Cells.

Protein-protein interactions (PPI) in cells play a pivotal role in cellular function and dynamics. Cellular proteostasis is maintained by PPI networks between molecular chaperones, co-chaperones, and client proteins. Consequently, strategies to visualize and analyze PPI in cells are useful in understanding protein homeostasis regulation. The Bimolecular Fluorescence Complementation (BiFC) assay has emerged as a useful tool for studying PPI between proteins in live or fixed cells. BiFC is based on the detection of fluorescence generated when interacting protein pairs, produced as fusion proteins with either the N- or C-terminal fragment of a fluorescent protein, are in sufficient proximity to permit reconstitution of the split fluorophore. Here, we describe the application of the BiFC assay to a model of chaperone-client interactions using Hsp90 and the validated client protein CDK4. This assay allows for the distribution and spatiotemporal analysis of HSP90-CDK4 complexes in live or fixed cells and is amenable to studying the effects of inhibitors and mutations on chaperone-client protein networks.

Complementation Assays for Co-chaperone Function.

The development of mutant microorganisms lacking J domain proteins (JDPs; formerly called Hsp40s) has enabled the development of complementation assays for testing the co-chaperone function of JDPs. In these assays, an exogenously expressed novel JDP is tested for its ability to functionally substitute for a non-expressed or nonfunctional endogenous JDP(s) by reversing a stress phenotype. For example, the in vivo functionality of prokaryotic JDPs can be tested on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant strain of the bacterium Escherichia coli (OD259). Similarly, the in vivo functionality of eukaryotic JDPs can be assessed in a thermosensitive ydj1 mutant strain of the yeast Saccharomyces cerevisiae (JJ160). Here we outline the use of these thermosensitive microorganisms in complementation assays to functionally characterize a JDP from the bacterium, Agrobacterium tumefaciens (AgtDnaJ), and a JDP from the trypanosomal parasite, Trypanosoma cruzi (TcJ2).

Optimized Microscale Protein Aggregation Suppression Assay: A Method for Evaluating the Holdase Activity of Chaperones.

Many molecular chaperones act as holdases by binding hydrophobic regions of substrates to prevent aggregation. Therefore, measuring holdase activity is an amenable method to determine chaperone activity. The holdase function is reliably and easily achieved by monitoring the suppression of heat-induced aggregation of well-characterized model protein substrates. However, the standard assay format requires large amounts of protein and hence is not applicable to all proteins. Using DnaK from Escherichia coli and heat-induced aggregation of malate dehydrogenase, we describe a protocol for absorbance and fluorescence-based miniaturized versions of the standard aggregation suppression assay that are affordable and have wide application for low abundance holdases. The assay can be used for both fundamental characterization of holdase function in proteins and screening of inhibitors of holdase activity.

Mutations F352A and Y528A in human HSP90α reduce fibronectin association and fibrillogenesis in cell-derived matrices.

HSP90 is a ubiquitously expressed chaperone protein that regulates the maturation of numerous substrate proteins called 'clients'. The glycoprotein fibronectin (FN) is an important protein of the extracellular matrix (ECM) and a client protein of HSP90. FN and HSP90 interact directly, and the FN ECM is regulated by exogenous HSP90 or HSP90 inhibitors. Here, we extend the analysis of the HSP90 - FN interaction. The importance of the N-terminal 70-kDa fragment of fibronectin (FN70) and FN type I repeat was demonstrated by competition for FN binding between HSP90 and the functional upstream domain (FUD) of the Streptococcus pyogenes F1 adhesin protein. Furthermore, His-HSP90α mutations F352A and Y528A (alone and in combination) reduced the association with full-length FN (FN-FL) and FN70 in vitro. Unlike wild type His-HSP90α, these HSP90 mutants did not enhance FN matrix assembly in the Hs578T cell line model when added exogenously. Interestingly, the HSP90 E353A mutation, which did not significantly reduce the HSP90 - FN interaction in vitro, dramatically blocked FN matrix assembly in Hs578T cell-derived matrices. Taken together, these data extend our understanding of the role of HSP90 in FN fibrillogenesis and suggest that promotion of FN ECM assembly by HSP90 is not solely regulated by the affinity of the direct interaction between HSP90 and FN.

Tenth International Symposium on the Hsp90 chaperone machine : Switzerland, October 19-23, 2022.

Hsp90 is a molecular chaperone responsible for regulating proteostasis under physiological and pathological conditions. Its central role in a range of diseases and potential as a drug target has focused efforts to understand its mechanisms and biological functions and to identify modulators that may form the basis for therapies. The 10th international conference on the Hsp90 chaperone machine was held in Switzerland in October 2022. The meeting was organized by Didier Picard (Geneva, Switzerland) and Johannes Buchner (Garching, Germany) with an advisory committee of Olivier Genest, Mehdi Mollapour, Ritwick Sawarkar, and Patricija van Oosten-Hawle. This was a much anticipated first in-person meeting of the Hsp90 community since 2018 after the COVID-19 pandemic led to the postponement of the 2020 meeting. The conference remained true to the tradition of sharing novel data ahead of publication, providing unparalleled depth of insight for both experts and newcomers to the field.

Comparative Analyses of Fucoidans from South African Brown Seaweeds That Inhibit Adhesion, Migration, and Long-Term Survival of Colorectal Cancer Cells.

Human colorectal cancer (CRC) is a recurrent, deadly malignant tumour with a high incidence. The incidence of CRC is of increasing alarm in highly developed countries, as well as in middle to low-income countries, posing a significant global health challenge. Therefore, novel management and prevention strategies are vital in reducing the morbidity and mortality of CRC. Fucoidans from South African seaweeds were hot water extracted and structurally characterised using FTIR, NMR and TGA. The fucoidans were chemically characterised to analyse their composition. In addition, the anti-cancer properties of the fucoidans on human HCT116 colorectal cells were investigated. The effect of fucoidans on HCT116 cell viability was explored using the resazurin assay. Thereafter, the anti-colony formation potential of fucoidans was explored. The potency of fucoidans on the 2D and 3D migration of HCT116 cells was investigated by wound healing assay and spheroid migration assays, respectively. Lastly, the anti-cell adhesion potential of fucoidans on HCT116 cells was also investigated. Our study found that Ecklonia sp. Fucoidans had a higher carbohydrate content and lower sulphate content than Sargassum elegans and commercial Fucus vesiculosus fucoidans. The fucoidans prevented 2D and 3D migration of HCT116 colorectal cancer cells to 80% at a fucoidan concentration of 100 µg/mL. This concentration of fucoidans also significantly inhibited HCT116 cell adhesion by 40%. Moreover, some fucoidan extracts hindered long-term colony formation by HCT116 cancer cells. In summary, the characterised fucoidan extracts demonstrated promising anti-cancer activities in vitro, and this warrants their further analyses in pre-clinical and clinical studies.

Biological and Medicinal Chemistry in Africa.

The young, fast-growing population of Africa means that harnessing the economic benefits of scientific research is critical to sustained and equitable growth in the continent. Moreover, the whole world would benefit from the added intellectual contribution that would come from nurturing African science. The high burden of neglected diseases in Africa makes chemical biology a particularly important field. In this editorial, the reconvergence of science conducted at the interface of chemistry and biology is placed in the context of African participation, its importance to global science and the unique blend of supporting and hindering factors that influence African scientific contributions. The new Biological and Medicinal Chemistry in Africa special collection showcases a broad spectrum of African chemical biology.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

Hsp70/Hsp90 Organising Protein (Hop): Coordinating Much More than Chaperones.

The Hsp70/Hsp90 organising protein (Hop, also known as stress-inducible protein 1/STI1/STIP1) has received considerable attention for diverse cellular functions in both healthy and diseased states. There is extensive evidence that intracellular Hop is a co-chaperone of the major chaperones Hsp70 and Hsp90, playing an important role in the productive folding of Hsp90 client proteins, although recent evidence suggests that eukaryotic Hop is regulatory within chaperone complexes rather than essential. Consequently, Hop is implicated in many key signalling pathways, including aberrant pathways leading to cancer. Hop is also secreted, and it is now well established that Hop interacts with the prion protein, PrPC, to mediate multiple signalling events. The intracellular and extracellular forms of Hop most likely represent two different isoforms, although the molecular determinants of these divergent functions are yet to be identified. There is also a growing body of research that reports the involvement of Hop in cellular activities that appear independent of either chaperones or PrPC. While the various cellular functions of Hop have been described, its biological function remains elusive. However, recent knockout studies in mammals suggest that Hop has an important role in embryonic development. This review provides a critical overview of the latest molecular, cellular and biological research on Hop, critically evaluating its function in healthy systems and how this function is adapted in diseased states.

CHIP: A Co-chaperone for Degradation by the Proteasome and Lysosome.

Protein homeostasis relies on a balance between protein folding and protein degradation. Molecular chaperones like Hsp70 and Hsp90 fulfill well-defined roles in protein folding and conformational stability via ATP-dependent reaction cycles. These folding cycles are controlled by associations with a cohort of non-client protein co-chaperones, such as Hop, p23, and Aha1. Pro-folding co-chaperones facilitate the transit of the client protein through the chaperone-mediated folding process. However, chaperones are also involved in proteasomal and lysosomal degradation of client proteins. Like folding complexes, the ability of chaperones to mediate protein degradation is regulated by co-chaperones, such as the C-terminal Hsp70-binding protein (CHIP/STUB1). CHIP binds to Hsp70 and Hsp90 chaperones through its tetratricopeptide repeat (TPR) domain and functions as an E3 ubiquitin ligase using a modified RING finger domain (U-box). This unique combination of domains effectively allows CHIP to network chaperone complexes to the ubiquitin-proteasome and autophagosome-lysosome systems. This chapter reviews the current understanding of CHIP as a co-chaperone that switches Hsp70/Hsp90 chaperone complexes from protein folding to protein degradation.

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.

Evolutionary progression of collective mutations in Omicron sub-lineages towards efficient RBD-hACE2: Allosteric communications between and within viral and human proteins.

The interaction between the Spike (S) protein of SARS-CoV-2 and the human angiotensin converting enzyme 2 (hACE2) is essential for infection, and is a target for neutralizing antibodies. Consequently, selection of mutations in the S protein is expected to be driven by the impact on the interaction with hACE2 and antibody escape. Here, for the first time, we systematically characterized the collective effects of mutations in each of the Omicron sub-lineages (BA.1, BA.2, BA.3 and BA.4) on both the viral S protein receptor binding domain (RBD) and the hACE2 protein using post molecular dynamics studies and dynamic residue network (DRN) analysis. Our analysis suggested that Omicron sub-lineage mutations result in altered physicochemical properties that change conformational flexibility compared to the reference structure, and may contribute to antibody escape. We also observed changes in the hACE2 substrate binding groove in some sub-lineages. Notably, we identified unique allosteric communication paths in the reference protein complex formed by the DRN metrics betweenness centrality and eigencentrality hubs, originating from the RBD core traversing the receptor binding motif of the S protein and the N-terminal domain of the hACE2 to the active site. We showed allosteric changes in residue network paths in both the RBD and hACE2 proteins due to Omicron sub-lineage mutations. Taken together, these data suggest progressive evolution of the Omicron S protein RBD in sub-lineages towards a more efficient interaction with the hACE2 receptor which may account for the increased transmissibility of Omicron variants.

Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition.

Contemporary medicinal chemistry considers fragment-based drug discovery (FBDD) and inhibition of protein-protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano-ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow-up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C-terminal domain. An in-silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose-dependent inhibitor of the target PPI.

Hsp90 and Associated Co-Chaperones of the Malaria Parasite.

Heat shock protein 90 (Hsp90) is one of the major guardians of cellular protein homeostasis, through its specialized molecular chaperone properties. While Hsp90 has been extensively studied in many prokaryotic and higher eukaryotic model organisms, its structural, functional, and biological properties in parasitic protozoans are less well defined. Hsp90 collaborates with a wide range of co-chaperones that fine-tune its protein folding pathway. Co-chaperones play many roles in the regulation of Hsp90, including selective targeting of client proteins, and the modulation of its ATPase activity, conformational changes, and post-translational modifications. Plasmodium falciparum is responsible for the most lethal form of human malaria. The survival of the malaria parasite inside the host and the vector depends on the action of molecular chaperones. The major cytosolic P. falciparum Hsp90 (PfHsp90) is known to play an essential role in the development of the parasite, particularly during the intra-erythrocytic stage in the human host. Although PfHsp90 shares significant sequence and structural similarity with human Hsp90, it has several major structural and functional differences. Furthermore, its co-chaperone network appears to be substantially different to that of the human host, with the potential absence of a key homolog. Indeed, PfHsp90 and its interface with co-chaperones represent potential drug targets for antimalarial drug discovery. In this review, we critically summarize the current understanding of the properties of Hsp90, and the associated co-chaperones of the malaria parasite.