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A multi-disciplinary Institute within the University of Oxford which focuses upon translational activities to catalyse the discovery of new medicines.
A systems-based approach to uterine fibroids identifies differential splicing associated with abnormal uterine bleeding.
BACKGROUND: Uterine fibroids (UFs), benign tumours prevalent in up to 80% of women of reproductive age, are associated with significant morbidity, including abnormal uterine bleeding, pain and infertility. Despite identification of key genomic alterations in MED12 and HMGA2, the pathogenic mechanisms underlying UFs and heavy menstrual bleeding (HMB) remain poorly understood. METHODS: To correlate systematically genetic, transcriptional and proteomic phenotypes, we conducted an integrative multi-omic approach utilising targeted DNA sequencing, RNA sequencing and proteomic methodologies, encompassing fibroid, myometrium, and endometrium tissues from 91 patients. RESULTS: In addition to confirming the presence of MED12 mutations, we identify variants in AHR and COL4A6. Multi-omic analysis of endometrium identifies latent factors that correlate with HMB and fibroid presence with driver mutations of MED12, AHR, and COL4A6, which are associated with pathways involved in angiogenesis, extracellular matrix organisation and RNA splicing. We propose a model, supported by in vivo evidence, where altered signalling of MED12-mutated fibroids influences RNA transcript isoform expression in endometrium, potentially leading to abnormal uterine bleeding. CONCLUSIONS: This study presents a comprehensive integrative approach, revealing that genetic alterations in UF may influence endometrial function via signalling impacts on the RNA splicing mechanism. Our findings advance the understanding of complex molecular pathways in UF pathogenesis and UF-associated endometrial dysfunction, offering insights for targeted therapeutic development.
Pleural fluid proteomics from patients with pleural infection shows signatures of diverse neutrophilic responses: The Oxford Pleural Infection Endotyping Study (TORPIDS-2).
BACKGROUND: Pleural infection is a complex disease with poor clinical outcomes and increasing incidence worldwide, yet its biological endotypes remain unknown. METHODS: We analysed 80 pleural fluid samples from the PILOT study, a prospective study on pleural infection, using unlabelled mass spectrometry. A total of 449 proteins were retained after filtering. Unsupervised hierarchical clustering and UMAP analyses were used to cluster samples and pathway analysis was performed to identify the biological processes. Protein signatures as identified by the pathway analysis were compared to microbiology as defined by 16S rRNA next generation sequencing. Spearman and exact Fischer's methods were used for correlation assessment. RESULTS: Higher neutrophil degranulation was correlated with increased glycolysis (OR=281, p<2.2E-16) and pentose phosphate activation (OR=371.45, p<2.2E-16). Samples dominated by Streptococcus pneumoniae exhibited higher neutrophil degranulation (OR=12.08, p=0.005), glycolysis (OR=11.4, p=0.006), and pentose phosphate activity (OR=12.82, p=0.004). On the other hand, samples dominated by anaerobes and Gram-negative bacteria exhibited lower neutrophil degranulation (OR=0.15, p=0.01, glycolysis (OR=0.14, p=0.01), and pentose phosphate activity (OR=0.07, p=0.001). Increased activity of the liver and retinoid X receptors (LXR-RXR) pathway was associated with lower risk of one-year mortality (OR=0.24, p=0.04). CONCLUSIONS: These findings suggest that pleural infection patients exhibit diverse responses of neutrophil mediated immunity, glycolysis, and pentose phosphate activation which are associated with microbiology. Therapeutic targeting of the LXR-RXR pathway with agonists is a possible treatment approach.
A new paradigm of islet adaptations in human pregnancy: insights from immunohistochemistry and proteomics.
Physiological changes during pregnancy support foetal growth, including adaptations in pancreatic islets to maintain glucose homeostasis. We investigate these adaptations using rare, high-quality pancreatic tissue from pregnant human donors and matched controls. We profile islets from pregnant donors using proteomics and assess α- and β-cell characteristics, as well as prolactin receptor and serotonin 2B receptor expression. Proteomic profiling of microdissected human islets identifies 7546 proteins but shows minimal differences in protein expression. In pregnancy, we show that islet area increases 1.9-fold, α- and β-cell areas increase 4.3- and 1.9-fold, driven by an increase in cell number rather than hypertrophy. Prolactin receptor expression is higher in α but not β cells, and serotonin 2B receptor is undetectable in β cells. Glucagon-like peptide-1 abundance increases 2.9-fold in α cells. These findings indicate that the molecular mechanisms driving pregnancy-induced islet adaptations in humans differ from those in mice, highlighting the need for human-based studies.
Protocol to profile spatially resolved NLRP3 inflammasome complexes using APEX2-based proximity labeling.
The NLRP3 inflammasome is a key multi-protein complex controlling inflammation, particularly interleukin-1β (IL-1β) production. Here, we present a protocol to profile spatially resolved NLRP3 inflammasome complexes using ascorbic peroxidase 2 (APEX2)-based proximity labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for design and generation of the fusion construct, characterization of the stable FLAG-NLRP3-APEX2 expression cell line by western blotting/imaging, biotinylated proteome enrichment, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Liang et al.1.
The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks.
DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognizes its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs is still not entirely clear. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain that we named the Ubiquitin Interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains in an avidity manner. SPRTN binding to ubiquitin chains via USD leads to ∼67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. In contrast, the constitutive components of the replisome during unperturbed or translesional DNA synthesis, namely proliferating cell nuclear antigen (PCNA) or monoUb-PCNA, respectively, were poorly degraded, if at all, by SPRTN. This study reveals that the poly-ubiquitination of DPCs serves as the key signal for SPRTN's rapid proteolysis and determines its substrate specificity towards DPCs, rather than the replisome.
Driving Therapeutic Innovation in Neurodegenerative Disease with Hydrogen Deuterium eXchange Mass Spectrometry.
Human neurodegenerative conditions such as Parkinson's and Alzheimer's Disease are characterized by the formation and deposition of toxic protein species which exacerbate neuronal dysfunction, impacting the structure and function of the healthy brain. Deciphering the mechanisms underlying protein (mis)folding and aggregation is not only essential for a more coherent view of neurodegeneration, but also crucial for the development of novel therapeutics targeting this family of disorders. Key pathological drivers of neurodegeneration, such as alpha-synuclein and tau proteins, have traditionally proved extremely challenging to characterize structurally due to their intrinsic and widespread structural plasticity. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) has emerged as a powerful tool to help circumvent this, owing to its ability to capture protein intrinsic disorder in solution, in addition to the transient structural conformations that typify protein aggregation pathways. This review brings together the most recent research where HDX-MS has shed light on mechanisms of neurodegeneration. We highlight how the technique has been successfully integrated into therapeutic development workflows targeting some of the most prevalent neurodegenerative diseases.
Amyloid-β disrupts APP-regulated protein aggregation and dissociation from recycling endosomal membranes.
Secretory proteins aggregate into non-soluble dense-core granules in recycling endosome-like compartments prior to regulated release. By contrast, aberrantly processed, secreted amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) form pathological extracellular amyloidogenic aggregations in late-stage Alzheimer's disease (AD). By examining living Drosophila prostate-like secondary cells, we show that both APP and Aβ peptides affect normal biogenesis of dense-core granules. These cells generate dense-core granules and secreted nanovesicles called Rab11-exosomes via evolutionarily conserved mechanisms within highly enlarged secretory compartments with recycling endosomal identity. The fly APP homologue, APP-like (APPL), associates with these vesicles and the compartmental limiting membrane, from where its extracellular domain modulates protein aggregation. Proteolytic release of this domain permits mini-aggregates to coalesce into a large central dense-core granule. Mutant Aβ expression disrupts this process and compartment motility, and increases aberrant lysosomal targeting, mirroring previously unexplained early-stage pathological events in AD. It also promotes cell-to-cell propagation of these endolysosomal defects, again phenocopying changes observed in AD. Our data therefore demonstrate physiological roles for APP in membrane-dependent protein aggregation, involving molecular mechanisms, which when disrupted by Aβ peptides, trigger Alzheimer's disease-relevant pathologies.
Pericardial and mediastinal fat-associated lymphoid clusters are rapidly activated in an alkane-induced model of systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is an autoimmune disease predominated by auto-antibodies that recognise cellular components. Pleural involvement is the most common SLE-related lung disease. Natural antibodies are rapidly secreted by innate-like B cells following perturbation of homeostasis and are important in the early stages of immune activation. The serous cavities are home to large numbers of innate-like B cells present both within serous fluid and resident within fat-associated lymphoid clusters (FALCs). FALCs are important hubs for B-cell activation and local antibody secretion within the body cavities. Patients with SLE can develop anti-phospholipid antibodies and in rare situations develop alveolar haemorrhage. Utilising delivery of the hydrocarbon oil pristane in C57BL/6 mice as a model of SLE we identify a rapid expansion of pleural cavity B cells as early as day 3 after intra-peritoneal pristane delivery. Following pristane delivery, pericardial B1 B cells are proliferative, express the plasma-cell surface marker CD138, and secrete both innate and class-switched antibodies highlighting that this cavity niche may play an unrecognised role in the initiation of lupus pleuritis.
Identification and characterization of human KALRN mRNA and Kalirin protein isoforms.
Kalirin is a multidomain protein with important roles in neurite outgrowth, and synaptic spine formation and remodeling. Genetic and pathophysiological links with various neuropsychiatric disorders associated with synaptic dysfunction and cognitive impairment have sparked interest in its potential as a pharmacological target. Multiple Kalirin proteoforms are detected in the adult human brain, yet we know little about the diversity of the transcripts that encode them or their tissue profiles. Here, we characterized full-length KALRN transcripts expressed in the adult human frontal lobe and hippocampus using rapid amplification of complementary DNA (cDNA) ends and nanopore long-read sequencing. For comparison with non-neural tissue, we also analyzed KALRN transcripts in the aorta. Multiple novel isoforms were identified and were largely similar between the two brain regions analyzed. Alternative splicing in the brain results in preferential inclusion of exon 37, which encodes 32 amino acids upstream of the second guanine nucleotide exchange factor (GEF) domain. Structural modeling predicts that a subset of these amino acids forms a conserved alpha helix. Although deletion of these amino acids had little effect on GEF activity, it did alter Kalirin-induced neurite outgrowth suggesting that this brain-enriched splicing event may be important for neural function. These data indicate that alternative splicing is potentially important for regulating Kalirin actions in the human brain.
Enabling equitable and affordable access to novel therapeutics for pandemic preparedness and response via creative intellectual property agreements.
The COVID-19 pandemic demonstrated that the current purely market-driven approaches to drug discovery and development alone are insufficient to drive equitable access to new therapies either in preparation for, or in response to, pandemics. A new global framework driven by equity is under negotiation at the World Health Organization to support pandemic preparedness and response. Some believe that the global intellectual property (IP) system itself is part of the problem and propose a purely Open Science approach. In this article, we discuss how existing IP frameworks and contractual agreements may be used to create rights and obligations to generate a more effective global response in future, drawing on experience gained in the COVID Moonshot program, a purely Open Science collaboration, and the ASAP AViDD drug discovery consortium, which uses a hybrid, phased model of Open Science, patent filing and contractual agreements. We conclude that 'straight to generic' drug discovery is appropriate in some domains, and that targeted patent protection, coupled with open licensing, can offer a route to generating affordable and equitable access for therapy areas where market forces have failed. The Extended Data contains a copy of our model IP policy, which can be used as a template by other discovery efforts seeking to ensure their drug candidates can be developed for globally equitable and affordable access.
SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids.
Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.
Protocol paper: a multi-center, double-blinded, randomized, 6-month, placebo-controlled study followed by 12-month open label extension to evaluate the safety and efficacy of Saracatinib in Fibrodysplasia Ossificans Progressiva (STOPFOP).
BACKGROUND: Fibrodysplasia Ossificans Progressiva (FOP) is a genetic, progressive and devastating disease characterized by severe heterotopic ossification (HO), loss of mobility and early death. There are no FDA approved medications. The STOPFOP team identified AZD0530 (saracatinib) as a potent inhibitor of the ALK2/ACVR1-kinase which is the causative gene for this rare bone disease. AZD0530 was proven to prevent HO formation in FOP mouse models. The STOPFOP trial investigates the repositioning of AZD0530, originally developed for ovarian cancer treatment, to treat patients with FOP. METHODS: The STOPFOP trial is a phase 2a study. It is designed as a European, multicentre, 6-month double blind randomized controlled trial of AZD0530 versus placebo, followed by a 12-month trial comparing open-label extended AZD0530 treatment with natural history data as a control. Enrollment will include 20 FOP patients, aged 18-65 years, with the classic FOP mutation (ALK2 R206H). The primary endpoint is objective change in heterotopic bone volume measured by low-dose whole-body computer tomography (CT) in the RCT phase. Secondary endpoints include 18F NaF PET activity and patient reported outcome measures. DISCUSSION: Clinical trials in rare diseases with limited study populations pose unique challenges. An ideal solution for limiting risks in early clinical studies is drug repositioning - using existing clinical molecules for new disease indications. Using existing assets may also allow a more fluid transition into clinical practice. With positive study outcome, AZD0530 may provide a therapy for FOP that can be rapidly progressed due to the availability of existing safety data from 28 registered clinical trials with AZD0530 involving over 600 patients. TRIAL REGISTRATION: EudraCT, 2019-003324-20. Registered 16 October 2019, https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-003324-20/NL . CLINICALTRIALS: gov , NCT04307953 . Registered 13 March 2020.
CACHE Challenge #2: Targeting the RNA Site of the SARS-CoV-2 Helicase Nsp13.
A critical assessment of computational hit-finding experiments (CACHE) challenge was conducted to predict ligands for the SARS-CoV-2 Nsp13 helicase RNA binding site, a highly conserved COVID-19 target. Twenty-three participating teams comprised of computational chemists and data scientists used protein structure and data from fragment-screening paired with advanced computational and machine learning methods to each predict up to 100 inhibitory ligands. Across all teams, 1957 compounds were predicted and were subsequently procured from commercial catalogs for biophysical assays. Of these compounds, 0.7% were confirmed to bind to Nsp13 in a surface plasmon resonance assay. The six best-performing computational workflows used fragment growing, active learning, or conventional virtual screening with and without complementary deep-learning scoring functions. Follow-up functional assays resulted in identification of two compound scaffolds that bound Nsp13 with a Kd below 10 μM and inhibited in vitro helicase activity. Overall, CACHE #2 participants were successful in identifying hit compound scaffolds targeting Nsp13, a central component of the coronavirus replication-transcription complex. Computational design strategies recurrently successful across the first two CACHE challenges include linking or growing docked or crystallized fragments and docking small and diverse libraries to train ultrafast machine-learning models. The CACHE #2 competition reveals how crowd-sourcing ligand prediction efforts using a distinct array of approaches followed with critical biophysical assays can result in novel lead compounds to advance drug discovery efforts.