Integrin-ligand interactions and cytokine production by monocytic cells

CD23 is a Type II glycoprotein that exists in both membrane-bound (mbCD23) and soluble (sCD23) forms and functions as the low affinity receptor for IgE. CD23 interacts with a range of proteins to fulfil its function in vivo. Through interactions with its ligands IgE and CD21, CD23 regulates the levels of IgE in serum. Soluble CD23 also interacts with members of the integrin family of membrane receptors. Integrins are heterodimeric transmembrane receptors that participate in bidirectional signalling across membranes and have a critical role in cellular adhesion reactions. CD23 interacts with four integrins, αVβ3 and αVβ5 from the αV integrin family, and αMβ2 and αXβ2, from the β2 family of integrins. Using peptide array technology, we identified set of overlapping peptides derived from the soluble CD23 sequence that interact with integrins expressed on the surface of monocytic cells and with purified αVβ3 and αVβ5 integrins in in vitro Biacore assays. These peptides all contained a common basic tripeptide motif, termed the Arg-Lys-Cys (RKC) motif. Integrins traditionally recognise and bind the Arg-Gly-Asp (RGD) tripeptide in their ligands in a cation-dependent manner. However, the in vitro interaction between RKC-containing peptides and purified integrins was determined to be cation-independent, salt-sensitive and independent of RGD binding. The interaction was blocked by full length CD23. Substitution of the residues in the RKC motif reduced or abolished binding of the peptides to integrins expressed on cells and in vitro, as measured by Biacore analysis, and abolished the competition with CD23. Taken together, these data suggest that the RKC motif is the site in CD23 that is recognised and bound by αVβ3 and αVβ5 integrins. The RKC motif can be considered a novel recognition motif for integrins, as it is cation-independent, and its binding is not blocked by the presence of RGD-containing integrin ligands. Therefore it is likely that the RKC motif interacts with integrins at a site other than that used for RGD-binding, similar to the interactions that have been described for the binding of the HIV Tat protein. The interaction between sCD23 and its integrin receptors is important in the regulation of cytokine production by monocytic cells. Most monocytic cells will express a combination of the different CD23-binding integrins simultaneously and, therefore, the cytokine output of that cell will be the net result of the interaction of CD23 with a combination of integrins. We used monoclonal antibodies to investigate the role of individual integrins in cytokine production. Antibodies were selected to allow comparision of the cytokine response between, a) integrin families, b) integrin subunits, and c) integrin epitopes. The cytokine profile induced by integrin ligation did not differ between the αV and β2 integrin families, although the concentration of cytokines produced varied depending on the heterodimer targeted. However, within a particular family, cytokine production induced by integrin ligation was specific and relied on the recognition of a precise epitope on the integrin. Cytokine production by CD23-binding integrins appears to require the ligation of the α subunit of the integrin heterodimer. We identified an antibody directed against the αVβ3 integrin that induced high levels of cytokine production. Cytokine production following ligation of the integrin with this antibody was dependent on activation of the ERK/MAPK pathway in cells. This production of cytokines and phosphorylation of ERK was enhanced by the addition of macrophage colony stimulating factor (M-CSF) and partially inhibited by an anti-TLR-2 antibody. Chronic stimulation (<3 days) of THP-1 cells with the anti-αVβ3 antibody in the presence of M-CSF led to morphological changes in the cell line associated with the development of a more macrophage-like phenotype and the continued production of cytokines. Analysis of the changes in cell surface marker expression and cytokine profiles suggested that the THP-1 cells had undergone an M2b programme of macrophage activation in response to αVβ3 ligation. Data presented herein reinforce the importance of the role of integrins in the control of adhesion-independent signalling pathways in suspension cells

Over-expression, purification and biochemical characterisation of trypanosomal heat shock protein

The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1

HSP90 interacts with the fibronectin N-terminal domains and increases matrix formation:

Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that controls the function and stability of a wide range of cellular client proteins. Fibronectin (FN) is an extracellular client protein of HSP90, and exogenous HSP90 or inhibitors of HSP90 alter the morphology of the extracellular matrix. Here, we further characterized the HSP90 and FN interaction. FN bound to the M domain of HSP90 and interacted with both the open and closed HSP90 conformations; and the interaction was reduced in the presence of sodium molybdate. HSP90 interacted with the N-terminal regions of FN, which are known to be important for matrix assembly

LRP1 is required for novobiocin-mediated fibronectin turnover:

Fibronectin (FN) plays a major role in the stability and organization of the extracellular matrix (ECM). We have previously demonstrated that FN interacts directly with Hsp90, as well as showing that the Hsp90 inhibitor novobiocin results in FN turnover via a receptor mediated process. However, the receptor involved has not been previously identified. LRP1 is a ubiquitous receptor responsible for the internalisation of numerous ligands that binds both Hsp90 and FN, and therefore we investigated whether LRP1 was involved in novobiocin-mediated FN turnover

In vitro analysis of putative cancer stem cell populations and chemosensitivity in the SW480 and SW620 colon cancer metastasis model:

The cancer stem cell (CSC) theory implicates a small subpopulation of cells with stem like properties, which is responsible for tumour initiation, development and metastasis. The unique biological and functional characteristics of CSCs, widely associated with treatment resistance, indicate an association between metastasis and stemness. It was hypothesised that metastatic cell lines may be enriched in CSCs and that this would correlate with a more resistant tumour. In the present study, the SW480 and SW620 paired cell lines derived from a colon adenocarcinoma and its lymph node metastasis, respectively were compared as an in vitro model of cancer progression. Their chemosensitivity and CSC properties were investigated. A range of in vitro assays were performed, including the side population assay, ALDEFLUOR assay, tumoursphere assay and assessment of CSC associated surface phenotypes

A combination approach in inhibiting type 2 diabetes-related enzymes using Ecklonia radiata fucoidan and acarbose

Although there are chemotherapeutic efforts in place for Type 2 diabetes mellitus (T2DM), there is a need for novel strategies (including natural products) to manage T2DM. Fucoidan, a sulphated polysaccharide was extracted from Ecklonia radiata. The integrity of the fucoidan was confirmed by structural analysis techniques such as FT-IR, NMR and TGA. In addition, the fucoidan was chemically characterised and tested for cell toxicity. The fucoidan was investigated with regards to its potential to inhibit -amylase and -glucosidase. The fucoidan was not cytotoxic and inhibited -glucosidase (IC50 19 g/mL) more strongly than the standard commercial drug acarbose (IC50 332 g/mL). However, the fucoidan lacked potency against -amylase. On the other hand, acarbose was a more potent inhibitor of -amylase (IC50 of 109 g/mL) than -glucosidase. Due to side effects associated with the use of acarbose, a combination approach using acarbose and fucoidan was investigated. The combination showed synergistic inhibition (>70%) of -glucosidase compared to when the drugs were used alone. The medicinal implication of this synergism is that a regimen with a reduced acarbose dose may be used, thus minimising side effects to the patient, while achieving the desired therapeutic effect for managing T2DM.The German Academic Exchange Service (DAAD) In-Region Scholarship; the South African National Research Foundation (NRF); Henderson Scholarship; Pearson- Young Memorial scholarship; the University of Pretoria; Rhodes University and KelpX.https://www.mdpi.com/journal/pharmaceuticsam2022BiochemistryGeneticsMicrobiology and Plant Patholog

Semi-synthesis and evaluation of sargahydroquinoic acid derivatives as potential antimalarial agents:

Malaria continues to present a major health problem, especially in developing countries. The development of new antimalarial drugs to counter drug resistance and ensure a steady supply of new treatment options is therefore an important area of research. Meroditerpenes have previously been shown to exhibit antiplasmodial activity against a chloroquinone sensitive strain of Plasmodium falciparum (D10). In this study we explored the antiplasmodial activity of several semi-synthetic analogs of sargahydroquinoic acid

Cytotoxic activity of marine sponge extracts from the sub-Antarctic Islands and the Southern Ocean

publisher versionOver the past 50 years, marine invertebrates, especially sponges, have proven to be a valuable source of new and/or bioactive natural products that have the potential to be further developed as lead compounds for pharmaceutical applications. Although marine benthic invertebrate communities occurring off the coast of South Africa have been explored for their biomedicinal potential, the natural product investigation of marine sponges from the sub-Antarctic Islands in the Southern Ocean for the presence of bioactive secondary metabolites has been relatively unexplored thus far. We report here the results for the biological screening of both aqueous and organic extracts prepared from nine specimens of eight species of marine sponges, collected from around Marion Island and the Prince Edward Islands in the Southern Ocean, for their cytotoxic activity against three cancer cell lines. The results obtained through this multidisciplinary collaborative research effort by exclusively South African institutions has provided an exciting opportunity to discover cytotoxic compounds from sub-Antarctic sponges, whilst contributing to our understanding of the biodiversity and geographic distributions of these cold-water invertebrates. Therefore, we acknowledge here the various contributions of the diverse scientific disciplines that played a pivotal role in providing the necessary platform for the future natural products chemistry investigation of these marine sponges from the sub- Antarctic Islands and the Southern Ocean. Significance: This study will contribute to understanding the biodiversity and geographic distributions of sponges in the Southern Ocean. This multidisciplinary project has enabled the investigation of marine sponges for the presence of cytotoxic compounds. Further investigation will lead to the isolation and identification of cytotoxic compounds present in the active sponge extracts.University of Cape Town; South African Medical Research Council; National Research Foundation (South Africa); CANSA; Rhodes University; Department of Science and Technology; Department of Environmental Affairs; SANA

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors