Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the ...dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.
Major histocompatibility complex class I molecules (MHC I) help protect jawed vertebrates by binding and presenting immunogenic peptides to cytotoxic T lymphocytes. Peptides are selected from a large ...diversity present in the endoplasmic reticulum. However, only a limited number of peptides complement the polymorphic MHC specificity determining pockets in a way that leads to high-affinity peptide binding and efficient antigen presentation. MHC I molecules possess an intrinsic ability to discriminate between peptides, which varies in efficiency between allotypes, but the mechanism of selection is unknown. Elucidation of the selection mechanism is likely to benefit future immune-modulatory therapies. Evidence suggests peptide selection involves transient adoption of alternative, presumably higher energy conformations than native peptide–MHC complexes. However, the instability of peptide-receptive MHC molecules has hindered characterization of such conformational plasticity. To investigate the dynamic nature of MHC, we refolded MHC proteins with peptides that can be hydrolyzed by UV light and thus released. We compared the resultant peptide-receptive MHC molecules with non-hydrolyzed peptide-loaded MHC complexes by monitoring the exchange of hydrogen for deuterium in solution. We found differences in hydrogen–deuterium exchange between peptide-loaded and peptide-receptive molecules that were negated by the addition of peptide to peptide-receptive MHC molecules. Peptide hydrolysis caused significant increases in hydrogen–deuterium exchange in sub-regions of the peptide-binding domain and smaller increases elsewhere, including in the α3 domain and the non-covalently associated β2-microglobulin molecule, demonstrating long-range dynamic communication. Comparing two MHC allotypes revealed allotype-specific differences in hydrogen–deuterium exchange, consistent with the notion that MHC I plasticity underpins peptide selection.
The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) ...cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFβ-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.
Abstract
BACKGROUND
Malignant paediatric nervous system tumours, such as medulloblastoma, ATRT and high-risk neuroblastoma commonly harbour tumour cells with stem-like features which are highly ...tumorigenic and resistant to conventional therapies. These tumours can exhibit high lethality and may result in severe sequelae that significantly affect paediatric patients' quality of life. Oncolytic virotherapy exploits viruses that preferentially infect and destroy tumour cells. These viruses present a unique advantage in targeting highly heterogeneous cancers as they possess a secondary mechanism of action, through which they induce an anti-tumoral immune response. The Zika virus (ZIKV) is capable of infecting and destroying aggressive human paediatric brain tumour and neuroblastoma cells in vitro. ZIKV effectively reduces brain tumour size in mice (xenograft model) and canines (naturally occurring) and can induce an immune response against canine brain tumours.
METHODS
Employing global expression omics profiling of ZIKV infection and mapping of viral protein-host protein interactions, we aim to elucidate the mechanisms which underpin ZIKVs therapeutic properties, both at the molecular and cellular pathway levels.
RESULTS
Through extensive transcriptome profiling of ZIKV-infected paediatric brain tumour, neuroblastoma and NPCs, we have identified a variety of pathways which are involved in the ZIKV oncolytic response in the tumour cells and its neuro-dysregulation of NPCs. Despite both brain tumour and neuroblastoma cells undergoing ZIKV-induced oncolysis, we observed there to be a heterogeneous response within these different tumour cells at the molecular level to lead to oncolysis. Additionally, the infected tumour cells demonstrate elevated immune system profiles which alludes to the immune response that ZIKV may raise within the patient’s body against the paediatric tumour. Analysing our findings alongside the neuro-dysregulation we observe in our ZIKV-infected NPCs is allowing us to build a safety profile for employing a ZIKV-based therapy, whilst contributing to the growing knowledge of Congenital ZIKV Syndrome.
The formation of protein coronae on nanoparticles (NPs) has been investigated almost exclusively in serum, despite the prevailing route of exposure being inhalation of airborne particles. In ...addition, an increasing number of nanomedicines, that exploit the airways as the site of delivery, are undergoing medical trials. An understanding of the effects of NPs on the airways is therefore required. To further this field, we have described the corona formed on polystyrene (PS) particles with different surface modifications and on titanium dioxide particles when incubated in human bronchoalveolar lavage fluid (BALF) from patients with pulmonary alveolar proteinosis (PAP). We show, using high-resolution quantitative mass spectrometry (MS
E
), that a large number of proteins bind with low copy numbers but that a few "core" proteins bind to all particles tested with high fidelity, averaging the surface properties of the different particles independent of the surface properties of the specific particle. The averaging effect at the particle surface means that differing cellular effects may not be due to the protein corona but due to the surface properties of the nanoparticle once inside the cell. Finally, the adherence of surfactant associated proteins (SP-A, B and D) suggests that there may be interactions with lipids and pulmonary surfactant (PSf), which could have potential in vivo health effects for people with chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), or those who have increased susceptibility toward other respiratory diseases.
Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly ...implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1–mediated epithelial–mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β–induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial–mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial–mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1–tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β–activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor–like repeats. Together, these data identify that aberrant bidirectional epithelial–mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.
Abstract
Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte ...antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-ω. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.
Oesophageal adenocarcinoma (OAC) has a relatively poor long‐term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, ...presented to cytotoxic T‐cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA‐I presented neoantigen from one patient, and report functional T‐cell responses from a predicted HLA‐II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T‐cell responses, emphasizing the need for improved strategies for neoantigen identification.
Oesophageal adenocarcinoma (OAC) has a relatively poor long‐term survival and limited treatment options. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA‐I presented neoantigen from one patient, and report functional T‐cell responses from a predicted HLA‐II neoantigen in a second patient containing both HLA I and II binding motifs.
Glycogen-specific kinase (GSK3beta) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3beta is implicated in ...many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3beta has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3beta kinase have been identified, the wider protein networks within which GSK3beta participates are not well known, and the consequences of these interactions not well understood. In this study, LC-MS/MS expression analysis was performed using knockout GSK3beta colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of beta-catenin. Consistent with the role of GSK3beta, we found that beta-catenin levels and canonical Wnt activity are unaffected by knockout of GSK3beta and therefore used this knockout cell model to identify other processes in which GSK3beta is implicated. Quantitative proteomic analysis revealed perturbation of proteins involved in cell-cell adhesion, and we characterized the phenotype and altered proteomic profiles associated with this. We also characterized the perturbation of metabolic pathways resulting from GSK3beta knockout and identified defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated beta-catenin we identified several of the diverse pathways and processes associated with GSK3beta function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is a critical epigenetic modification that is established and maintained across the genome by DNA methyltransferase enzymes (Dnmts). Altered patterns of DNA methylation are a frequent ...occurrence in many tumor genomes, and inhibitors of Dnmts have become important epigenetic drugs. Azacitidine is a cytidine analog that is incorporated into DNA and induces the specific inhibition and proteasomal-mediated degradation of Dnmts. The downstream effects of azacitidine on CpG methylation and on gene transcription have been widely studied in many systems, but how azacitidine impacts the proteome is not well-understood. In addition, with its specific ability to induce the rapid degradation of Dnmts (in particular, the primary maintenance DNA methyltransferase, Dnmt1), it may be employed as a specific chemical knockdown for investigating the Dnmt1-associated functional or physical interactome. In this study, we use quantitative proteomics to analyze the degradation profile of proteins in the nuclear proteome of cells treated with azacitidine. We identify specific proteins as well as multiple pathways and processes that are impacted by azacitidine. The Dnmt1 interaction partner, Uhrf1, exhibits significant azacitidine-induced degradation, and this azacitidine-induced degradation is independent of the levels of Dnmt1 protein. We identify multiple other chromatin- and epigenetic-associated factors, including the bromodomain-containing transcriptional regulator, Brd2. We show that azacitidine induces highly specific perturbations of the Dnmt1-associated proteome, and while interaction partners such as Uhrf1 are sensitive to azacitidine, others such as the Dnmt1 interaction partner and stability regulator, Usp7, are not. In summary, we have conducted the first comprehensive proteomic analysis of the azacitidine-sensitive nuclear proteome, and we show how 5-azacitidine can be used as a specific probe to explore Dnmt- and chromatin-related protein networks.
PDF
