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.

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.

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.

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.

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.

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.

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 as a regulator of extracellular matrix dynamics.

The extracellular matrix (ECM) is a dynamic and organised extracellular network assembled from proteins and carbohydrates exported from the cell. The ECM is critical for multicellular life, providing spatial and temporal cellular cues to maintain tissue homeostasis. Consequently, ECM production must be carefully balanced with turnover to ensure homeostasis; ECM dysfunction culminates in disease. Hsp90 is a molecular chaperone central to protein homeostasis, including in the ECM. Intracellular and extracellular Hsp90 isoforms collaborate to regulate the levels and status of proteins in the ECM via multiple mechanisms. In so doing, Hsp90 regulates ECM dynamics, and changes in Hsp90 levels or activity support the development of ECM-related diseases, like cancer and fibrosis. Consequently, Hsp90 levels may have prognostic value, while inhibition of Hsp90 may have therapeutic potential in conditions characterised by ECM dysfunction.

General Structural and Functional Features of Molecular Chaperones.

Molecular chaperones are a group of structurally diverse and highly conserved ubiquitous proteins. They play crucial roles in facilitating the correct folding of proteins in vivo by preventing protein aggregation or facilitating the appropriate folding and assembly of proteins. Heat shock proteins form the major class of molecular chaperones that are responsible for protein folding events in the cell. This is achieved by ATP-dependent (folding machines) or ATP-independent mechanisms (holders). Heat shock proteins are induced by a variety of stresses, besides heat shock. The large and varied heat shock protein class is categorised into several subfamilies based on their sizes in kDa namely, small Hsps (HSPB), J domain proteins (Hsp40/DNAJ), Hsp60 (HSPD/E; Chaperonins), Hsp70 (HSPA), Hsp90 (HSPC), and Hsp100. Heat shock proteins are localised to different compartments in the cell to carry out tasks specific to their environment. Most heat shock proteins form large oligomeric structures, and their functions are usually regulated by a variety of cochaperones and cofactors. Heat shock proteins do not function in isolation but are rather part of the chaperone network in the cell. The general structural and functional features of the major heat shock protein families are discussed, including their roles in human disease. Their function is particularly important in disease due to increased stress in the cell. Vector-borne parasites affecting human health encounter stress during transmission between invertebrate vectors and mammalian hosts. Members of the main classes of heat shock proteins are all represented in Plasmodium falciparum, the causative agent of cerebral malaria, and they play specific functions in differentiation, cytoprotection, signal transduction, and virulence.

Fucoidan Structure and Its Impact on Glucose Metabolism: Implications for Diabetes and Cancer Therapy.

Fucoidans are complex polysaccharides derived from brown seaweeds which consist of considerable proportions of L-fucose and other monosaccharides, and sulphated ester residues. The search for novel and natural bioproduct drugs (due to toxicity issues associated with chemotherapeutics) has led to the extensive study of fucoidan due to reports of it having several bioactive characteristics. Among other fucoidan bioactivities, antidiabetic and anticancer properties have received the most research attention in the past decade. However, the elucidation of the fucoidan structure and its biological activity is still vague. In addition, research has suggested that there is a link between diabetes and cancer; however, limited data exist where dual chemotherapeutic efforts are elucidated. This review provides an overview of glucose metabolism, which is the central process involved in the progression of both diseases. We also highlight potential therapeutic targets and show the relevance of fucoidan and its derivatives as a candidate for both cancer and diabetes therapy.

Coumarin-Annulated Ferrocenyl 1,3-Oxazine Derivatives Possessing In Vitro Antimalarial and Antitrypanosomal Potency.

A tailored series of coumarin-based ferrocenyl 1,3-oxazine hybrid compounds was synthesized and investigated for potential antiparasitic activity, drawing inspiration from the established biological efficacy of the constituent chemical motifs. The structural identity of the synthesized compounds was confirmed by common spectroscopic techniques: NMR, HRMS and IR. Biological evaluation studies reveal that the compounds exhibit higher in vitro antiparasitic potency against the chemosensitive malarial strain (3D7 P. falciparum) over the investigated trypanosomiasis causal agent (T. b. brucei 427) with mostly single digit micromolar IC50 values. When read in tandem with the biological performance of previously reported structurally similar non-coumarin, phenyl derivatives (i.e., ferrocenyl 1,3-benzoxazines and α-aminocresols), structure-activity relationship analyses suggest that the presence of the coumarin nucleus is tolerated for biological activity though this may lead to reduced efficacy. Preliminary mechanistic studies with the most promising compound (11b) support hemozoin inhibition and DNA interaction as likely mechanistic modalities by which this class of compounds may act to produce plasmocidal and antitrypanosomal effects.

Hop depletion reduces HSF1 levels and activity and coincides with reduced stress resilience.

Heat-shock factor 1 (HSF1) regulates the transcriptional response to stress and controls expression of molecular chaperones required for cell survival. Here we report that HSF1 is regulated by the abundance of the Hsp70-Hsp90 organizing protein (Hop/STIP1). HSF1 levels were significantly reduced in Hop-depleted HEK293T cells. HSF1 transcriptional activity at the Hsp70 promoter, and binding of a biotinylated HSE oligonucleotide under both basal and heat-shock conditions were significantly reduced. Hop-depleted HEK293T cells were more sensitive to the HSF1 inhibitor KRIBB11 and showed reduced short-term proliferation, and reduced long-term survival under basal and heat-shock conditions. HSF1 nuclear localization was reduced in response to heat-shock and the nuclear staining pattern in Hop-depleted cells was punctate. Taken together, these data suggest that Hop regulates HSF1 function under both basal and stress conditions through a mechanism involving changes in levels, activity and subcellular localization, and coincides with reduced cellular fitness.

The in Vitro Antiplasmodial and Antiproliferative Activity of New Ferrocene-Based α-Aminocresols Targeting Hemozoin Inhibition and DNA Interaction.

The conjugation of organometallic complexes to known bioactive organic frameworks is a proven strategy revered for devising new drug molecules with novel modes of action. This approach holds great promise for the generation of potent drug leads in the quest for therapeutic chemotypes with the potential to overcome the development of clinical resistance. Herein, we present the in vitro antiplasmodial and antiproliferative investigation of ferrocenyl α-aminocresol conjugates assembled by amalgamation of the organometallic ferrocene unit and an α-aminocresol scaffold possessing antimalarial activity. The compounds pursued in the study exhibited higher toxicity towards the chemosensitive (3D7) and -resistant (Dd2) strains of the Plasmodium falciparum parasite than to the human HCC70 triple-negative breast cancer cell line. Indication of cross-resistance was absent for the compounds evaluated against the multi-resistant Dd2 strain. Structure-activity analysis revealed that the phenolic hydroxy group and rotatable σ bond between the α-carbon and NH group of the α-amino-o-cresol skeleton are crucial for the biological activity of the compounds. Spectrophotometric techniques and in silico docking simulations performed on selected derivatives suggest that the compounds show a dual mode of action involving hemozoin inhibition and DNA interaction via minor-groove binding. Lastly, compound 9 a, identified as a possible lead, exhibited preferential binding for the plasmodial DNA isolated from 3D7 P. falciparum trophozoites over the mammalian calf thymus DNA, thereby substantiating the enhanced antiplasmodial activity of the compounds. The presented research demonstrates the strategy of incorporating organometallic complexes into known biologically active organic scaffolds as a viable avenue to fashion novel multimodal compounds with potential to counter the development drug resistance.

Use of a non-hepatic cell line highlights limitations associated with cell-based assessment of metabolically induced toxicity.

Metabolically induced drug-toxicity is a major cause of drug failure late in drug optimization phases. Accordingly, in vitro metabolic profiling of compounds is being introduced at earlier stages of the drug discovery pipeline. An increasingly common method to obtain these profiles is through overexpression of key CYP450 metabolic enzymes in immortalized liver cells, to generate competent hepatocyte surrogates. Enhanced cytotoxicity is presumed to be due to toxic metabolite production via the overexpressed enzyme. However, metabolically induced toxicity is a complex multi-parameter phenomenon and the potential background contribution to metabolism arising from the use of liver cells which endogenously express CYP450 isoforms is consistently overlooked. In this study, we sought to reduce the potential background interference by applying this methodology in kidney-derived HEK293 cells which lack endogenous CYP450 expression. Overexpression of CYP3A4 resulted in increased HEK293 proliferation, while exposure to four compounds with reported metabolically induced cytotoxicity in liver-derived cells overexpressing CYP3A4 resulted in no increase in cytotoxicity. Our results indicate that overexpression of a single CYP450 isoform in hepatic cell lines may not be a reliable method to discriminate which enzymes are responsible for metabolic induced cytotoxicity.

STIP1/HOP Regulates the Actin Cytoskeleton through Interactions with Actin and Changes in Actin-Binding Proteins Cofilin and Profilin.

Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.

Novobiocin-ferrocene conjugates possessing anticancer and antiplasmodial activity independent of HSP90 inhibition.

A series of tailored novobiocin-ferrocene conjugates was prepared in moderate yields and investigated for in vitro anticancer and antiplasmodial activity against the MDA-MB-231 breast cancer line and Plasmodium falciparum 3D7 strain, respectively. While the target compounds displayed moderate anticancer activity against the breast cancer cell line with IC50 values in the mid-micromolar range, compounds 10a-c displayed promising antiplasmodial activity as low as 0.889 µM. Furthermore, the most promising compounds were tested for inhibitory effects against a postulated target, heat shock protein 90 (Hsp90). A selection of tailored novobiocin derivatives bearing the organometallic ferrocene unit were synthesized and characterized by common spectroscopic techniques. The target compounds were investigated for in vitro anticancer and antimalarial activity against the MDA-MB-231 breast cancer cell line and Plasmodium falciparum 3D7 strain, respectively.

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Hop/STIP1 depletion alters nuclear structure via depletion of nuclear structural protein emerin.

Hop/STIP1 is a co-chaperone of Hsp70 and Hsp90 that regulates a number of cell biology processes via interactions with cellular proteins. Here we report a new relationship between Hop and the nuclear structural protein emerin in maintenance of nuclear morphology. Depletion or overexpression of Hop resulted in the reduction of emerin protein levels via proteasomal and lysosomal pathways. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which could accommodate Hsp70 but not Hsp90, and that TPR2AB is required for the association. Loss of Hop or emerin led to a deformation of nuclear structure, a statistically significant decrease in nuclear size, and was associated with changes in the levels of nuclear proteins, lamin A-C and fibrillarin. The nuclear defects upon Hop loss could be rescued by emerin overexpression. Taken together, these data suggest that Hop stabilises emerin and that loss of Hop alters nuclear structure via emerin degradation.

Heterodimer formation by Oct4 and Smad3 differentially regulates epithelial-to-mesenchymal transition-associated factors in breast cancer progression.

The multifunctional cytokine TGF-ß crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-ß and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-ß in late stages compared to early stages of the disease. In vitro studies showed that TGF-ß elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-ß-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-ß, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-ß signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients.

Extracellular Hsp90 and TGFß regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model.

BACKGROUND: Tumour metastasis remains the major cause of death in cancer patients and, to date, the mechanism and signalling pathways governing this process are not completely understood. The TGF-ß pathway is the most commonly mutated pathway in cancer, however its role in cancer progression is controversial as it can function as both a promoter and a suppressor of metastasis. Although previous studies have suggested a role for the molecular chaperone Hsp90 in regulating the TGF-ß pathway, the level at which this occurs as well as the consequences in terms of colon cancer metastasis are unknown. METHODS: The paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and its lymph node metastasis, respectively, were used as an in vitro model to study key cellular processes required for metastasis. The status of the TGF-ß pathway was examined in these cells using ELISA, flow cytometry, western blot analysis and confocal microscopy. Furthermore, the effect of addition or inhibition of the TGF-ß pathway and Hsp90 on adhesion, migration and anchorage-independent growth, was determined in the cell lines. RESULTS: When comparing the canonical TGF-ß1 pathway in the genetically paired cell lines our data suggests that this pathway may be constitutively active in the SW620 metastasis-derived cell line and not the SW480 primary tumour-derived line. In addition, we report that, when present in combination, TGF-ß1 and Hsp90ß stimulate anchorage-independent growth, reduce adhesion and stimulate migration. This effect is potentiated by inhibition of the TGF-ß1 receptor and occurs via an alternate TGF-ß1 pathway, mediated by αvß6 integrin. Interestingly, in the SW620 cells, activation of this alternate TGF-ß1 signalling machinery does not appear to require inhibition of the canonical TGF-ß1 receptor, which would allow them to respond more effectively to the pro-metastasis stimulus of a combination of Hsp90ß and TGF-ß1 and this could account for the increased migratory capacity of these cells. CONCLUSIONS: In this study we report an apparent synergy between TGF-ß1 and Hsp90ß in stimulating migratory behaviour of colon cancer cells when signalling occurs via αvß6 integrin as opposed to the canonical TGF-ß1 pathway.