MRC Toxicology Unit

SUMMARYModification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1400 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3100 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

Aging is a major risk factor for neurodegeneration and is characterized by diverse cellular and molecular hallmarks. To understand the origin of these hallmarks, we studied the effects of aging on the transcriptome, translatome, and proteome in the brain of short-lived killifish. We identified a cascade of events in which aberrant translation pausing led to altered abundance of proteins independently of transcriptional regulation. In particular, aging caused increased ribosome stalling and widespread depletion of proteins enriched in basic amino acids. These findings uncover a potential vulnerable point in the aging brain's biology-the biogenesis of basic DNA and RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity, proteostasis, and the biosynthesis of macromolecules.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Abstract Increased protein synthesis drives proliferation of established tumours. However, how mRNA translation contributes to early tumorigenesis remains unclear. Following oncogene activation, hepatocytes enter a senescent phase characterized by deposition of extracellular matrix (ECM) niches which promote subsequent progression to hepatocellular carcinoma (HCC). We demonstrate an inverse relationship between translation rates and transition from this oncogene-induced senescent state to proliferating HCC. We have found that this is due to tight coupling of the mRNA translation rate at the endoplasmic reticulum (ER) and lysosomal degradation of membrane/secretory proteins. Specifically, increased routing of newly synthesized proteins from the ER alters endosomal trafficking of membrane integrins towards late endosomes/lysosomes which, in turn, opposes generation of ECM-rich tumour initiation niches. Consistently, inhibition of protein synthesis during early tumorigenesis accelerates HCC initiation, challenging the current thinking on the relationship between mRNA translation rates and cancer. Our results also highlight that the administration of current therapeutics designed to suppress translation rates may need to be carefully timed to improve patient outcomes.

BackgroundCampylobacter jejuni and Campylobacter coli are the major causative agents of bacterial gastroenteritis worldwide and are known obligate microaerophiles. Despite being sensitive to oxygen and its reduction products, both species are readily isolated from animal food products kept under atmospheric conditions where they face high oxygen tension levels.ResultsIn this study, Transposon Directed Insertion-site Sequencing (TraDIS) was used to investigate the ability of one C. jejuni strain and two C. coli strains to overcome oxidative stress, using H2O2 to mimic oxidative stress. Genes were identified that were required for oxidative stress resistance for each individual strain but also allowed a comparison across the three strains. Mutations in the perR and ahpC genes were found to increase Campylobacter tolerance to H2O2. The roles of these proteins in oxidative stress were previously known in C. jejuni, but this data indicates that they most likely play a similar role in C. coli. Mutation of czcD decreased Campylobacter tolerance to H2O2. The role of CzcD, which functions as a zinc exporter, has not previously been linked to oxidative stress. The TraDIS data was confirmed using defined deletions of perR and czcD in C. coli 15-537360.ConclusionsThis is the first study to investigate gene fitness in both C. jejuni and C. coli under oxidative stress conditions and highlights both similar roles for certain genes for both species and highlights other genes that have a role under oxidative stress.

Mesothelioma is an aggressive cancer of the mesothelial layer associated with an extensive fibrotic response. The latter is in large part mediated by cancer-associated fibroblasts which mediate tumour progression and poor prognosis. However, understanding of the crosstalk between cancer cells and fibroblasts in this disease is mostly lacking. Here, using co-cultures of patient-derived mesothelioma cell lines and lung fibroblasts, we demonstrate that fibroblast activation is a self-propagated process producing a fibrotic extracellular matrix (ECM) and triggering drug resistance in mesothelioma cells. Following characterisation of mesothelioma cells/fibroblasts signalling crosstalk, we identify several FDA-approved targeted therapies as far more potent than standard-of-care Cisplatin/Pemetrexed in ECM-embedded co-culture spheroid models. In particular, the SRC family kinase inhibitor, Saracatinib, extends overall survival well beyond standard-of-care in a mesothelioma genetically-engineered mouse model. In short, we lay the foundation for the rational design of novel therapeutic strategies targeting mesothelioma/fibroblast communication for the treatment of mesothelioma patients.

Patients with Human Hyper IgM syndromes (HIGM) developed pulmonary and gastrointestinal infections since infancy and most patients have mutations in the CD40 ligand (CD40L) gene. Most HIGM patients compared to healthy subjects have higher/similar IgM and lower IgG, and IgA serum concentrations but gut antibody concentrations are unknown. CD40L on activated T-cells interacts with CD40 on B-cells, essential for the formation of germinal centres (GCs) inside secondary lymphoid organs (SLOs), where high-affinity antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, are produced. C57BL6-CD40 ligand deficient mice (C57BL6-cd40l -/-), are a model of HIGM, because serum immunoglobulin concentrations parallel levels observed in HIGM patients and have higher faecal IgA concentrations. In mice, TGFβ and other cytokines induce IgA production. To compare and evaluate B-cell populations and IgA-producing plasma cells in peritoneal lavage, non-gut-associated SLOs, spleen/inguinal lymph nodes (ILN), and gut-associated SLOs, mesenteric lymph nodes (MLN)/Peyer´s patches (PP) of unimmunised C57BL6-cd40l -/- and C57BL6-wild-type (WT)mice. Peritoneal lavages, spleens, ILN, MLN, and PP from 8-10 weeks old C57BL6-cd40l -/- and WT mice, were obtained. Organ cryosections were analysed by immunofluorescence and B-cell populations and IgA-positive plasma cell suspensions by flow cytometry. In unimmunised WT mice, GCs were only observed in the gut-associated SLOs, but GCs were absent in all C57BL6-cd40l -/- SLOs. PP and MLN of C57BL6-cd40l -/- mice exhibited a significantly higher number of IgA-producing cells than WT mice. In the spleen and ILN of C57BL6-cd40l- /- mice IgA-producing cells significantly decreased, while IgM-positive plasma cells increased. C57BL6-cd40l -/- B-1 cells were more abundant in all analysed SLOs, whereas in WT mice most B-1 cells were contained within the peritoneal cavity. C57BL6-cd40l -/- B-cells in MLN expressed a higher TGFβ receptor-1 than WT mice. Mouse strains small intestine microvilli (MV), have a similar frequency of IgA-positive cells. Together our results confirm the role of PP and MLN as gut inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites even in the absence of GCs. IgA antibodies play a pivotal role in neutralising, eliminating, and regulating potential pathogens and microorganisms in the gut.

SummaryAlpha-synuclein (aSYN) is a small synaptic protein that is linked to Parkinson’s disease (PD) and other synucleinopathies. aSYN has been shown to undergo protein phase separation. We found that aSYN phase separation, in vitro and in cells, is regulated by vesicle-associated membrane protein 2 (VAMP2), the vesicular SNARE protein. We show that aSYN phase separation is promoted by C-terminal electrostatic interaction, which in cells is mediated via interaction with VAMP2. The condensate formation is specific for the R-SNARE VAMP2 and critically dependent on aSYN’s capacity to bind to lipid membranes. In vitro, the addition of VAMP2 decreases the saturation concentration of aSYN phase separation, which is mediated by its juxtamembrane domain. Our results delineate a molecular mechanism for the regulation of aSYN phase separation, indicating a potential switch from the dispersed to the phase-separated state during vesicle cycling.HighlightsAlpha-synuclein phase separation is regulated by electrostatic interactionsVAMP2 initiates alpha-synuclein condensation in cellsPositive charges within the juxtamembrane domain of VAMP2 initiate alpha-synuclein phase separation in vitroAlpha-synuclein condensates nucleate on lipid membranes and assemble vesicular structures in cells

Abstract Tyre wear is an increasing source of ambient particulate matter pollution in the UK and around the world. Currently, tyre wear accounts for 11% of total particulate matter (PM2.5) in the UK, and over the next 10 years that figure is projected to steadily increase, with UK brake and tyre emissions projected to reach 6 kilotonnes by 2030. Being able to reliably identify and monitor tyre wear PM in ambient sources is of increasing importance due to its potential human health effects. Here we present a series of characterisation experiments on tyre particles, size-fractionated using a dynamometer. Chemical and elemental composition has been determined by inductively coupled plasma mass spectrometry (ICP-MS) and two dimensional gas chromatography time of flight mass spectrometry (GCxGC-TOF-MS), with particle morphology studied using a scanning electron microscope and energy dispersive spectroscopy (SEM-EDS) and an electrical low pressure impactor (ELPI). The potential health effects of these particles have then been assessed in vitro using human bronchial epithelial cells following the IARC characteristics of carcinogenicity, specifically looking at (pro)inflammatory cytokines, cytotoxicity and mitochondrial function. Tyre wear particles have the ability to reduce mitochondrial function significantly from doses as low as 5μg/ml after 24 hours, with the induction of cytokines and chemokines currently being profiled. Additionally, a representative elemental and chemical fingerprint of tyre rubber has now been established. By understanding the composition and morphology of these particles, we can begin to detect them in atmospheric samples, and using in vitro models, understand their potential health effects.

Parkinson’s disease (PD) is characterised by selective death of dopaminergic (DA) neurons in the midbrain and motor function impairment. Gastrointestinal issues often precede motor deficits in PD, indicating that the gut-brain axis is involved in the pathogenesis of this disease. The features of PD include both mitochondrial dysfunction and activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER). PINK1 is a mitochondrial kinase involved in the recycling of defective mitochondria, and PINK1 mutations cause early-onset PD. Like PD patients, pink1 mutant Drosophila show degeneration of DA neurons and intestinal dysfunction. These mutant flies also lack vital proteins due to sustained activation of the kinase R-like endoplasmic reticulum kinase (dPerk), a kinase that induces the UPR. Here, we investigated the role of dPerk in intestinal dysfunction. We showed that intestinal expression of dPerk impairs mitochondrial function, induces cell death, and decreases lifespan. We found that suppressing dPerk in the intestine of pink1-mutant flies rescues intestinal cell death and is neuroprotective. We conclude that in a fly model of PD, blocking gut-brain transmission of UPR-mediated toxicity, is neuroprotective.